Genes and uses for plant improvement

ABSTRACT

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. Exemplary recombinant DNA expresses a succinate semialdehyde dehydrogenase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priority to U.S. application Ser. No. 11/982,700, filed Nov. 1, 2007, which application is a divisional of U.S. application Ser. No. 11/188,298, filed Jul. 22, 2005, which claims the benefit of priority to U.S. Provisional Application No. 60/592,978, filed Jul. 31, 2004, which applications are incorporated herein by reference and made a part hereof in their entirety, and the benefit of priority of each of which is claimed herein.

INCORPORATION OF SEQUENCE LISTING

Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-ROMs, each containing the file named “1379958.txt”, which is 70,686,720 bytes (measured in MS-WINDOWS) and was created on Oct. 21, 2014, are herein incorporated by reference.

FIELD OF THE INVENTION

Disclosed herein are inventions in the field of plant genetics and developmental biology. More specifically, this invention provides transgenic seeds for crops, wherein the genome of said seed comprises recombinant DNA, the expression of which results in the production of transgenic plants that have improved trait(s).

BACKGROUND OF THE INVENTION

Transgenic plants with improved traits, such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of genes that are useful in recombinant DNA constructs for production of transformed plants with improved properties.

SUMMARY OF THE INVENTION

This invention provides transgenic seeds, transgenic plants and DNA constructs with trait-improving recombinant DNA from a gene for a protein having an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group consisting of SEQ ID NO: 270 and its homologs through SEQ ID NO: 538, where the respective homolog proteins have amino acid sequences SEQ ID NO: 539 through SEQ ID NO: 22568, as indicated in Table 17. In some cases of trait improvement, the recombinant DNA encodes a protein; in other cases, the recombinant DNA suppresses endogenous protein expression. In a broad aspect this invention provides transgenic seeds for growing crop plants with improved traits, such crop plants with improved traits and the plant parts including transgenic seed produced by such crop plants. The improved traits provided by the recombinant DNA in the transgenic crop plant of this invention are identified by comparison to a control plant, i.e., a plant without the trait-improving recombinant DNA. In one aspect of the invention, transgenic crop plant grown from the transgenic seed has improved yield, as compared to the yield of a control plant, e.g., a plant without the recombinant DNA that produces the increased yield. Some plants of this invention exhibit increased yield by producing a yield increase under non-stress conditions. Other plants of this invention exhibit increased yield by producing a yield increase under one or more environmental stress conditions including, but not limited to, water deficit stress, cold stress, heat stress, high salinity stress, shade stress, and low nitrogen availability stress. Still other plants of this invention have other improved phenotypes, such as improved plant development, plant morphology, plant physiology or seed composition as compared to a corresponding trait of a control plant. The various aspects of this invention are especially useful for transgenic seed and transgenic plants having improved traits in corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

The invention also comprises recombinant DNA constructs. In one aspect, such recombinant DNA constructs useful for the transgenic seed and transgenic plants of this invention comprise a promoter functional in a plant cell operably linked to a DNA segment for expressing a protein associated with a trait in a model plant or a homologue. In another aspect the recombinant DNA constructs useful for the transgenic seed and transgenic plants of this invention comprise a promoter functional in a plant cell operably linked to a DNA segment for suppressing the level of an endogenous plant protein which is a homologue to a model-plant protein, the suppression of which is associated with an improved trait. Suppression can be effected by any of a variety of methods known in the art, e.g., post transcriptional suppression by anti-sense, sense, dsRNA and the like or by transcriptional suppression.

This invention also provides a method of producing a transgenic crop plant having at least one improved trait, wherein the method comprises providing to a grower of transgenic seeds comprising recombinant DNA for expression or suppression of a trait-improving gene provided herein, and growing transgenic plant from said transgenic seed. Such methods are used to generate transgenic crop plants having at least one improved trait under one or more environmental stress conditions including, but not limited to, water deficit stress, cold stress, heat stress, high salinity stress, shade stress, and low nitrogen availability stress. In another aspect, such methods are used to generate transgenic crop plants having improved plant development, plant morphology, plant physiology or seed component phenotype as compared to a corresponding phenotype of a control plant. Of particular interest are uses of such methods to generate transgenic crop plants having increased yield under non-stress condition, or under one or more stress conditions.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides transgenic plant seed having in its genome trait-improving recombinant DNA and transgenic plants grown from such seed which exhibit an improved trait as compared a control plant. In one aspect, the invention provides transgenic plants where the improved trait is one or more of improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity. Particular transgenic plants grown from transgenic seeds of this invention exhibit increased seed yield. Recombinant DNA constructs used in this invention comprise recombinant DNA disclosed herein which produces mRNA to modulate gene expression imparting improved traits to plants.

“Gene” means all or part of the DNA that encodes a protein or mRNA, e.g., chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA, and includes DNA regions flanking the coding sequences, e.g., introns, 5′UTR, 3′UTR, promoters and other DNA involved in the regulation of expression.

“Transgenic seed” means plant seed having a genome altered by the incorporation of recombinant DNA, e.g., by transformation. “Transgenic plant” means a plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains the recombinant DNA in its genome. “Recombinant DNA” means a DNA molecule having a genetically engineered modification introduced through a combination of endogenous and/or exogenous DNA elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. Recombinant DNA can exist outside of a cell, e.g., as a PCR fragment or in a plasmid, or can be integrated into a genome such as a plant genome.

“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances the characteristic is visible to the human eye, e.g., seed or plant size, or can be measured by biochemical techniques, e.g., detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.

“Control plant” is a plant without trait-improving recombinant DNA. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. One suitable control plant is a non-transgenic plant of the parental line that was used to generate a transgenic plant. Another suitable control plant is a transgenic plant that comprises recombinant DNA without the specific trait producing DNA, e.g., simply a marker gene. Another suitable control plant is a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, e.g., in field conditions, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used, i.e., a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, i.e., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.

“Trait improvement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait improvement is measured quantitatively. For example, the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait improvement is only measured qualitatively. It is known that there are natural variations in a trait. Therefore, the trait improvement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait improvement includes, but not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include improved plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.

“Yield-limiting environment” means a condition under which a plant would have the limitation on yield including environmental stress conditions.

“Stress condition” means a condition unfavorable for a plant, which adversely affects plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” means sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) is one physiological measure of plant water deficit. RWC measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. RWC can result from heat, drought, high salinity and induced osmotic stress.

“Cold stress” means exposure of a plant to temperatures below, e.g., at least two or more degrees Celsius below, those temperatures that are normal for a particular species or particular strain of plant.

“Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives enough amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. “Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. “Ammonium” means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc. Those skilled in the art know what constitutes such soil, media and fertilizer inputs for most plant species. “Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition; a useful low nitrogen availability stress is a growth condition with 50% or less of the conventional nitrogen inputs.

“Shade stress” means a limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress is exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few other examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre.

“Increased yield” of a transgenic plant of this invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, corn yield is measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield is often achieved from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA is used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.

“Expression” means transcription of DNA to produce RNA. The resulting RNA includes mRNA encoding a protein, antisense RNA that is complementary to an mRNA encoding a protein, or an RNA transcript comprising a combination of sense and antisense gene regions, such as for use in RNAi gene suppression. Expression also means production of encoded protein from mRNA.

“Promoter” means a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. “Tissue preferred” promoters preferentially regulate expression in certain tissues, such as leaves, roots, or seeds. “Tissue specific” promoters predominately regulate expression only in certain tissues. “Cell type” specific promoter primarily regulate expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. “Inducible” and “repressible” promoters regulate expression under environmental influences, under the effect of anaerobic conditions, certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute a class of “non-constitutive” promoters. “Constitutive” promoters are promoters which are active under most conditions. “Anti-sense orientation” refers to a DNA sequence that is operably linked to a promoter in an orientation where the anti-sense strand is transcribed. “Operably linked” refers to an association of two or more DNA elements in a single construct so that the function of one is affected by the other. For example, a promoter is operably linked with transcribable DNA when it is capable of affecting the expression of that DNA; that is, the coding DNA is under the transcriptional control of the promoter.

“Consensus sequence” means an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.

“Homologs” means genes that produce functionally similar proteins, e.g., in the same organism or in different organisms. A gene can be related to a homolog gene by descent from a common ancestral DNA. Homologs include genes where the relationship is by speciation, e.g., often called orthologs, or by genetic duplication, e.g., often called paralogs. More specifically, “orthologs” include homologs in different species that evolved from a common ancestral gene by specification. Normally orthologs retain the same function in the course of evolution. “Paralogs” include homologs in the same species that have diverged from each other as a consequence of genetic duplication.

“Percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of sequences is the number of identical components which are shared divided by the total number of sequence components in the segment used as a reference over a window of alignment which is the smaller of the sequences. “Percent identity” (“% identity”) is the identity fraction times 100. “% identity” to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.

“Arabidopsis” means plants of Arabidopsis thaliana.

Recombinant DNA Constructs

This invention provides recombinant DNA constructs comprising DNA elements for imparting one or more improved traits to transgenic plant. Such constructs typically comprise a promoter operatively linked to DNA to provide for expression of a protein or RNA for gene suppression in a target plant. Recombinant DNA constructs can also include additional regulatory elements, such as 5′ or 3′ untranslated regions (UTRs) such as polyadenylation sites, introns, and transit or signal peptides. Such recombinant DNA constructs are assembled using methods known to those of ordinary skill in the art.

In certain embodiments, recombinant DNA constructs comprise sense-oriented, trait-imparting DNA operably linked to a promoter that is functional in a plant to provide for expression of the trait-imparting DNA in the sense orientation such that a desired protein is produced. In other embodiments at least a part of the trait-imparting DNA is in an anti-sense orientation for gene suppression activity.

Recombinant DNA constructs, especially for expressing proteins are typically prepared with a 3′ UTR that a polyadenylation site and signal. Recombinant DNA constructs can also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference.

Table 1 provides a list of genes that can provide trait-imparting DNA for recombinant DNA constructs. DNA from each gene was used in a model plant (Arabidopsis) to discover associations with improved traits. The DNA was also used to identify homologs from which a consensus amino acid sequence is defined for characterizing the aspects of the invention where recombinant DNA is incorporated in the transgenic seeds, transgenic plants, DNA constructs and methods of this invention. With reference to Table 1:

“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing. “PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA “construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA. “gene” refers to an arbitrary name used to identify the particular DNA. “orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter. “species” refers to the organism from which the particular DNA was derived.

TABLE 1 Nuc SEQ ID Pep SEQ ID construct_id Gene orientation Species 1 270 14324 CGPG1560 SENSE Arabidopsis thaliana 2 271 17484 CGPG2630 SENSE Arabidopsis thaliana 3 272 19109 CGPG1381 ANTI-SENSE Arabidopsis thaliana 4 273 70423 CGPG3165 SENSE Arabidopsis thaliana 5 274 70424 CGPG3180 SENSE Arabidopsis thaliana 6 275 70480 CGPG3833 SENSE Arabidopsis thaliana 7 276 70509 CGPG2420 SENSE Arabidopsis thaliana 8 277 70647 CGPG4334 SENSE Arabidopsis thaliana 9 278 70675 CGPG4519 SENSE Arabidopsis thaliana 10 279 70829 CGPG518 SENSE Arabidopsis thaliana 11 280 70849 CGPG596 SENSE Arabidopsis thaliana 12 281 71627 CGPG1270 SENSE Arabidopsis thaliana 13 282 71934 CGPG2294 SENSE Arabidopsis thaliana 14 283 72615 CGPG4829 SENSE Arabidopsis thaliana 15 284 72927 CGPG1477 SENSE Arabidopsis thaliana 16 285 73014 CGPG5692 SENSE Xenorhabdus nematophilus 85816 17 286 73559 CGPG6535 SENSE Bacillus subtilis 168 18 287 74251 CGPG5489 SENSE Arabidopsis thaliana 19 288 19631 CGPG3627 SENSE Arabidopsis thaliana 20 289 70121 CGPG2380 SENSE Saccharomyces cerevisiae 21 290 70654 CGPG4352 SENSE Arabidopsis thaliana 22 291 70696 CGPG4590 SENSE Arabidopsis thaliana 23 292 70713 CGPG1462 ANTI-SENSE Arabidopsis thaliana 24 293 70740 CGPG3700 SENSE Arabidopsis thaliana 25 294 71321 CGPG4418 SENSE Arabidopsis thaliana 26 295 71835 CGPG4634 SENSE Arabidopsis thaliana 27 296 72934 CGPG5798 SENSE Saccharomyces cerevisiae 28 297 72945 CGPG5787 SENSE Saccharomyces cerevisiae 29 298 72980 CGPG5773 SENSE Saccharomyces cerevisiae 30 299 73504 CGPG6480 SENSE Synechocystis sp. PCC 6803 31 300 73507 CGPG6504 SENSE Bacillus subtilis 168 32 301 73573 CGPG6462 SENSE Agrobacterium tumefacians C58 33 302 73586 CGPG6471 SENSE Bacillus subtilis 168 34 303 73770 CGPG5435 SENSE Arabidopsis thaliana 35 304 74105 CGPG6574 SENSE Xenorhabdus nematophilus 86068 36 305 74111 CGPG6622 SENSE Escherichia coli K-12 37 306 74136 CGPG6632 SENSE Synechocystis 38 307 74139 CGPG6561 SENSE Escherichia coli K-12 39 308 74267 CGPG5364 SENSE Arabidopsis thaliana 40 309 74291 CGPG5363 SENSE Arabidopsis thaliana 41 310 74318 CGPG5826 SENSE Arabidopsis thaliana 42 311 74319 CGPG5831 SENSE Arabidopsis thaliana 43 312 74324 CGPG5885 SENSE Arabidopsis thaliana 44 313 74512 CGPG32 SENSE Arabidopsis thaliana 45 314 74583 CGPG6649 SENSE Ralstonia metallidurans CH34 46 315 70427 CGPG3067 SENSE Arabidopsis thaliana 47 316 71811 CGPG4426 SENSE Arabidopsis thaliana 48 317 73463 CGPG6384 SENSE Ralstonia metallidurans CH34 49 318 72081 CGPG5279 SENSE Glycine max 50 319 10139 CGPG101 ANTI-SENSE Arabidopsis thaliana 51 320 11410 CGPG103 SENSE Arabidopsis thaliana 52 321 11604 CGPG48 ANTI-SENSE Arabidopsis thaliana 53 322 12368 CGPG1006 SENSE Arabidopsis thaliana 54 323 13502 CGPG1354 SENSE Arabidopsis thaliana 55 324 13745 CGPG1576 ANTI-SENSE Arabidopsis thaliana 56 325 13821 CGPG1569 SENSE Arabidopsis thaliana 57 326 14240 CGPG1697 SENSE Arabidopsis thaliana 58 327 14718 CGPG1082 SENSE Arabidopsis thaliana 59 328 17022 CGPG1774 SENSE Arabidopsis thaliana 60 329 17924 CGPG2882 SENSE Arabidopsis thaliana 61 330 18259 CGPG3368 SENSE Arabidopsis thaliana 62 331 19171 CGPG2952 SENSE Saccharomyces cerevisiae 63 332 19201 CGPG2332 SENSE Arabidopsis thaliana 64 333 19317 CGPG3662 SENSE Xanthomonas 65 334 70417 CGPG3427 SENSE Arabidopsis thaliana 66 335 70467 CGPG3785 SENSE Arabidopsis thaliana 67 336 70806 CGPG712 SENSE Arabidopsis thaliana 68 337 70818 CGPG479 SENSE Arabidopsis thaliana 69 338 70820 CGPG655 SENSE Arabidopsis thaliana 70 339 70919 CGPG4029 SENSE Glycine max 71 340 71623 CGPG4696 SENSE Arabidopsis thaliana 72 341 71662 CGPG4679 SENSE Glycine max 73 342 71693 CGPG4652 SENSE Glycine max 74 343 72384 CGPG4639 SENSE Saccharomyces cerevisiae 75 344 72439 CGPG5075 SENSE Arabidopsis thaliana 76 345 72619 CGPG4835 SENSE Arabidopsis thaliana 77 346 72624 CGPG4842 SENSE Arabidopsis thaliana 78 347 72715 CGPG5521 SENSE Saccharomyces cerevisiae 79 348 72754 CGPG5548 SENSE Saccharomyces cerevisiae 80 349 72819 CGPG4989 SENSE Arabidopsis thaliana 81 350 75516 CGPG7689 SENSE Glycine max 82 351 75701 CGPG7856 SENSE Glycine max 83 352 73515 CGPG6473 SENSE Bacillus subtilis 168 84 353 74684 CGPG6360 SENSE Arabidopsis thaliana 85 354 19542 CGPG3069 SENSE Arabidopsis thaliana 86 355 19618 CGPG3574 SENSE Arabidopsis thaliana 87 356 19649 CGPG3140 SENSE Arabidopsis thaliana 88 357 19745 CGPG3973 SENSE Glycine max 89 358 19768 CGPG4096 SENSE Glycine max 90 359 19772 CGPG3939 SENSE Glycine max 91 360 19779 CGPG4113 SENSE Glycine max 92 361 19833 CGPG4074 SENSE Glycine max 93 362 19862 CGPG3961 SENSE Glycine max 94 363 19879 CGPG4009 SENSE Glycine max 95 364 70445 CGPG3728 SENSE Arabidopsis thaliana 96 365 70738 CGPG3195 SENSE Arabidopsis thaliana 97 366 71437 CGPG4043 SENSE Glycine max 98 367 71572 CGPG4520 SENSE Arabidopsis thaliana 99 368 71617 CGPG1227 SENSE Arabidopsis thaliana 100 369 72532 CGPG4780 SENSE Arabidopsis thaliana 101 370 72757 CGPG5572 SENSE Arabidopsis thaliana 102 371 73412 CGPG6448 SENSE Pseudomonas syringae var tomato DC3000 103 372 74102 CGPG6550 SENSE Bacillus halodurans C-125 104 373 72633 CGPG4853 SENSE Arabidopsis thaliana 105 374 72456 CGPG4745 SENSE Arabidopsis thaliana 106 375 72963 CGPG1746 SENSE Arabidopsis thaliana 107 376 70426 CGPG3199 SENSE Arabidopsis thaliana 108 377 70772 CGPG4627 SENSE Arabidopsis thaliana 109 378 71137 CGPG125 SENSE Arabidopsis thaliana 110 379 71529 CGPG2808 SENSE Arabidopsis thaliana 111 380 71601 CGPG1858 SENSE Arabidopsis thaliana 112 381 72362 CGPG983 SENSE Arabidopsis thaliana 113 382 72466 CGPG4767 SENSE Arabidopsis thaliana 114 383 72524 CGPG4770 SENSE Arabidopsis thaliana 115 384 73085 CGPG5689 SENSE Synechocystis sp. PCC 6803 116 385 74241 CGPG5457 SENSE Arabidopsis thaliana 117 386 74247 CGPG5475 SENSE Arabidopsis thaliana 118 387 74284 CGPG5413 SENSE Arabidopsis thaliana 119 388 74652 CGPG6168 SENSE Arabidopsis thaliana 120 389 70437 CGPG3706 SENSE Arabidopsis thaliana 121 390 71633 CGPG857 SENSE Arabidopsis thaliana 122 391 72948 CGPG5617 SENSE Arabidopsis thaliana 123 392 72519 CGPG4749 SENSE Arabidopsis thaliana 124 393 10475 CGPG399 SENSE Arabidopsis thaliana 125 394 11120 CGPG459 ANTI-SENSE Arabidopsis thaliana 126 395 19736 CGPG4129 SENSE Glycine max 127 396 71606 CGPG4715 SENSE Arabidopsis thaliana 128 397 71840 CGPG4353 SENSE Arabidopsis thaliana 129 398 74240 CGPG5454 SENSE Arabidopsis thaliana 130 399 74331 CGPG5834 SENSE Arabidopsis thaliana 131 400 74610 CGPG6048 SENSE Arabidopsis thaliana 132 401 75527 CGPG7682 SENSE Glycine max 133 402 70681 CGPG4584 SENSE Arabidopsis thaliana 134 403 71663 CGPG4638 SENSE Xanthomonas 135 404 72769 CGPG5573 SENSE Arabidopsis thaliana 136 405 71508 CGPG1541 SENSE Arabidopsis thaliana 137 406 74248 CGPG5476 SENSE Arabidopsis thaliana 138 407 72771 CGPG2166 SENSE Arabidopsis thaliana 139 408 72085 CGPG5228 SENSE Arabidopsis thaliana 140 409 72744 CGPG5563 SENSE Saccharomyces cerevisiae 141 410 73039 CGPG810 SENSE Arabidopsis thaliana 142 411 73054 CGPG5754 SENSE Saccharomyces cerevisiae 143 412 73501 CGPG6456 SENSE Agrobacterium tumefacians C58 144 413 19707 CGPG4179 SENSE Glycine max 145 414 19951 CGPG3941 SENSE Glycine max 146 415 19967 CGPG4032 SENSE Glycine max 147 416 70543 CGPG3815 SENSE Arabidopsis thaliana 148 417 70707 CGPG1273 ANTI-SENSE Arabidopsis thaliana 149 418 70719 CGPG1712 ANTI-SENSE Arabidopsis thaliana 150 419 71134 CGPG817 SENSE Arabidopsis thaliana 151 420 71146 CGPG2928 SENSE Arabidopsis thaliana 152 421 71660 CGPG4690 SENSE Arabidopsis thaliana 153 422 72086 CGPG5236 SENSE Arabidopsis thaliana 154 423 72632 CGPG4852 SENSE Arabidopsis thaliana 155 424 72716 CGPG5529 SENSE Saccharomyces cerevisiae 156 425 72723 CGPG1848 SENSE Arabidopsis thaliana 157 426 72987 CGPG1787 SENSE Arabidopsis thaliana 158 427 74109 CGPG6606 SENSE Xenorhabdus nematophilus 86068 159 428 74140 CGPG6569 SENSE Bacillus halodurans C-125 160 429 74191 CGPG6597 SENSE Rhodobacter sphaeroides 2.4.1 161 430 74265 CGPG5356 SENSE Arabidopsis thaliana 162 431 74369 CGPG6076 SENSE Arabidopsis thaliana 163 432 70217 CGPG6 SENSE Arabidopsis thaliana 164 433 72711 CGPG1846 SENSE Arabidopsis thaliana 165 434 70932 CGPG4089 SENSE Glycine max 166 435 73518 CGPG6497 SENSE Pseudomonas fluorescens PfO-1 167 436 19771 CGPG4011 SENSE Glycine max 168 437 73549 CGPG6460 SENSE Xenorhabdus nematophilus 85816 169 438 72994 CGPG5803 SENSE Saccharomyces cerevisiae 170 439 71928 CGPG1617 SENSE Arabidopsis thaliana 171 440 72903 CGPG5584 SENSE Arabidopsis thaliana 172 441 73017 CGPG5733 SENSE Saccharomyces cerevisiae 173 442 74587 CGPG6774 SENSE Agrobacterium tumefacians C58 174 443 72453 CGPG4735 SENSE Arabidopsis thaliana 175 444 72967 CGPG5742 SENSE Saccharomyces cerevisiae 176 445 72961 CGPG5591 SENSE Arabidopsis thaliana 177 446 73070 CGPG5627 SENSE Arabidopsis thaliana 178 447 73475 CGPG6385 SENSE Rhodopseudomonas palustris CGA009 179 448 72916 CGPG1814 SENSE Arabidopsis thaliana 180 449 72969 CGPG5789 SENSE Saccharomyces cerevisiae 181 450 74449 CGPG6659 SENSE Agrobacterium tumefaciens 182 451 16615 CGPG2539 SENSE Agrobacterium 183 452 19187 CGPG3310 SENSE Arabidopsis thaliana 184 453 19648 CGPG3134 SENSE Arabidopsis thaliana 185 454 70354 CGPG3995 SENSE Glycine max 186 455 70421 CGPG2942 SENSE Arabidopsis thaliana 187 456 70459 CGPG3758 SENSE Arabidopsis thaliana 188 457 70465 CGPG3775 SENSE Arabidopsis thaliana 189 458 70683 CGPG4587 SENSE Arabidopsis thaliana 190 459 70725 CGPG2097 ANTI-SENSE Arabidopsis thaliana 191 460 70852 CGPG1465 SENSE Arabidopsis thaliana 192 461 71112 CGPG934 SENSE Arabidopsis thaliana 193 462 71127 CGPG945 SENSE Arabidopsis thaliana 194 463 71132 CGPG1561 SENSE Arabidopsis thaliana 195 464 71217 CGPG95 SENSE Arabidopsis thaliana 196 465 71645 CGPG4688 SENSE Arabidopsis thaliana 197 466 71726 CGPG3894 SENSE Arabidopsis thaliana 198 467 72432 CGPG4562 SENSE Arabidopsis thaliana 199 468 72450 CGPG4732 SENSE Arabidopsis thaliana 200 469 72455 CGPG4742 SENSE Arabidopsis thaliana 201 470 72727 CGPG5522 SENSE Saccharomyces cerevisiae 202 471 72817 CGPG4987 SENSE Arabidopsis thaliana 203 472 72992 CGPG5777 SENSE Saccharomyces cerevisiae 204 473 73007 CGPG5760 SENSE Saccharomyces cerevisiae 205 474 73073 CGPG5688 SENSE Synechocystis sp. PCC 6803 206 475 73506 CGPG6496 SENSE Pseudomonas fluorescens PfO-1 207 476 74107 CGPG6590 SENSE Sinorhizobium meliloti 1021 208 477 74117 CGPG6575 SENSE Xenorhabdus nematophilus 86068 209 478 74131 CGPG6592 SENSE Synechocystis sp. PCC 6803 210 479 74344 CGPG5929 SENSE Arabidopsis thaliana 211 480 14320 CGPG1229 SENSE Arabidopsis thaliana 212 481 16756 CGPG2117 SENSE Arabidopsis thaliana 213 482 17448 CGPG2673 SENSE Arabidopsis thaliana 214 483 17633 CGPG2839 SENSE Arabidopsis thaliana 215 484 18876 CGPG3096 SENSE Arabidopsis thaliana 216 485 19120 CGPG1976 ANTI-SENSE Arabidopsis thaliana 217 486 19221 CGPG2958 SENSE Arabidopsis thaliana 218 487 70206 CGPG4116 SENSE Glycine max 219 488 70223 CGPG53 SENSE Arabidopsis thaliana 220 489 70347 CGPG3147 SENSE Arabidopsis thaliana 221 490 70406 CGPG1687 SENSE Arabidopsis thaliana 222 491 70469 CGPG3791 SENSE Arabidopsis thaliana 223 492 70564 CGPG1864 SENSE Arabidopsis thaliana 224 493 70601 CGPG2917 SENSE Arabidopsis thaliana 225 494 70612 CGPG3721 SENSE Arabidopsis thaliana 226 495 70720 CGPG1358 ANTI-SENSE Arabidopsis thaliana 227 496 70735 CGPG2661 SENSE Arabidopsis thaliana 228 497 70846 CGPG377 SENSE Arabidopsis thaliana 229 498 70923 CGPG4020 SENSE Glycine max 230 499 71149 CGPG3457 SENSE Arabidopsis thaliana 231 500 71608 CGPG4687 SENSE Arabidopsis thaliana 232 501 71739 CGPG4345 SENSE Arabidopsis thaliana 233 502 72014 CGPG5230 SENSE Arabidopsis thaliana 234 503 72051 CGPG5241 SENSE Arabidopsis thaliana 235 504 74259 CGPG5343 SENSE Arabidopsis thaliana 236 505 72463 CGPG4760 SENSE Arabidopsis thaliana 237 506 72902 CGPG5597 SENSE Arabidopsis thaliana 238 507 74572 CGPG6640 SENSE Synechocystis 239 508 73055 CGPG5768 SENSE Saccharomyces cerevisiae 240 509 74103 CGPG6558 SENSE Escherichia coli K-12 241 510 72921 CGPG5781 SENSE Saccharomyces cerevisiae 242 511 72968 CGPG5772 SENSE Saccharomyces cerevisiae 243 512 19703 CGPG4172 SENSE Glycine max 244 513 19946 CGPG4097 SENSE Glycine max 245 514 19980 CGPG3914 SENSE Glycine max 246 515 70435 CGPG3701 SENSE Arabidopsis thaliana 247 516 71114 CGPG1657 SENSE Arabidopsis thaliana 248 517 72451 CGPG4733 SENSE Arabidopsis thaliana 249 518 72947 CGPG5607 SENSE Glycine max 250 519 73012 CGPG5786 SENSE Saccharomyces cerevisiae 251 520 73022 CGPG5622 SENSE Arabidopsis thaliana 252 521 73488 CGPG6394 SENSE Bacillus subtilis 168 253 522 73901 CGPG5237 SENSE Arabidopsis thaliana 254 523 73964 CGPG5804 SENSE Saccharomyces cerevisiae 255 524 74019 CGPG5706 SENSE Bacillus subtilis 168 256 525 74022 CGPG5724 SENSE Arabidopsis thaliana 257 526 74114 CGPG6551 SENSE Agrobacterium tumefacians C58 258 527 74262 CGPG5353 SENSE Arabidopsis thaliana 259 528 74292 CGPG5367 SENSE Arabidopsis thaliana 260 529 74302 CGPG5384 SENSE Arabidopsis thaliana 261 530 74325 CGPG5898 SENSE Arabidopsis thaliana 262 531 74429 CGPG6689 SENSE Bacillus subtilis 168 263 532 74440 CGPG6682 SENSE Bacillus halodurans C-125 264 533 74462 CGPG6668 SENSE Synechocystis 265 534 74465 CGPG6692 SENSE Bacillus subtilis 168 266 535 74474 CGPG6669 SENSE Synechocystis 267 536 74505 CGPG6783 SENSE Escherichia coli K-12 268 537 74507 CGPG6799 SENSE Xenorhabdus nematophilus 85816 269 538 74562 CGPG6764 SENSE Bacillus subtilis 168

Recombinant DNA

Trait-imparting DNA for use in this invention for improved traits in plants is disclosed herein as having a DNA sequence of SEQ ID NO:1 through SEQ ID NO:269 and any of the respective homologs. A subset of the trait-imparting DNA includes fragments with less than the full DNA sequence, e.g., consisting of oligonucleotides of at least about 15 to 20 or more consecutive nucleotides from one of the disclosed sequences. Such oligonucleotides are fragments of the larger molecules having a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 269, and find use, for example as probes and primers for detection of the polynucleotides of the invention or for cloning DNA for use in this invention.

Useful DNA includes variants of the disclosed DNA. Such variants include naturally occurring, including homologous DNA from genes of the same or a different species, or non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one nucleotide of a disclosed DNA without causing the amino acid sequence of the protein produced to be changed. Hence, useful DNA can have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.

Homologs of the trait-imparting DNA generally demonstrate significant identity with the DNA provided herein. Homologous DNA is substantially identical to a trait-imparting DNA if, when the nucleotide sequences are optimally aligned there is at least about 60% nucleotide identity, or higher, e.g., at least 70% or 80% or 85% or even 90% identity or higher, such as 95% or 98% identity over a comparison window of at least 50 to 100 nucleotides, and up to the entire length of the trait-imparting DNA. Optimal alignment of sequences for aligning a comparison window can be conducted by algorithms including computerized implementations of the algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference DNA sequence can represent a full-length coding sequence or a portion.

Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 270 through SEQ ID NO: 538, as well as homologs of such proteins.

One method to identify homologs of the proteins useful in this invention is by comparison of the amino acid sequence of the trait-imparting protein to amino acid sequences of proteins from the same or different organisms, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. In one method a local sequence alignment program, e.g., BLAST, is used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) is used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to described proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 539 through SEQ ID NO: 22568 to the proteins with amino acid sequences of SEQ ID NO: 270 through SEQ ID NO: 538 is found is found in Table 17.

Aspects of the invention also use DNA encoding functional homolog proteins which differ in one or more amino acids from those of protein encoded by disclosed trait-imparting DNA as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence are selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence are selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention uses DNA encoding proteins that differ in one or more amino acids from those of protein encoded from a described trait-imparting DNA as the result of deletion or insertion of one or more amino acids in a native sequence.

Homologs of the proteins encoded by disclosed trait-improving DNA will generally demonstrate significant sequence identity, e.g., at least 50% amino acid sequence identity or higher such as at least 70% identity or at least 80% or at least 90% identity with an amino acid sequence of SEQ ID NO:270 through SEQ ID NO:538. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence of a protein encoded by a disclosed trait-imparting DNA and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO:270 through SEQ ID NO:538.

Genes that are homologs to each other can be grouped into families and included in multiple sequence alignments to allow a consensus sequence to be derived. This analysis enables the derivation of conserved and class- (family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. A consensus sequence is used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship and the corresponding trait-imparting DNA. Thus, this invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence.

Promoters

Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant DNA to provide for expression of desired genes in transgenic plant cells.

It is well known in the art that promoters are usefully altered to contain multiple “enhancer sequences” to assist in elevating gene expression. By including an enhancer sequence with such constructs, expression is generally enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, and can also be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, 5′ enhancing elements are introns. Particularly useful enhancers are the 5′ introns of the rice actin 1 gene and the rice actin 2 gene. Other enhancers include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.

In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of causing sufficient expression in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Such water-deficit-inducible promoters are disclosed in U.S. 2004-0123347 A1, incorporated herein by reference.

In other aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell, et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger, et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy, et al., (1996) Plant Mol Biol. 31(6):1205-1216).

In still other aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).

Gene Overexpression

“Gene overexpression” means expression, e.g., of a gene at a level in its native host that exceeds levels of expression in a non-transgenic host. In many embodiments of the invention, a recombinant DNA construct provides gene overexpression, e.g., as identified in Table 1.

Gene Suppression

Gene suppression includes any of the well-known methods for suppressing expression, typically indicated by reduced levels of protein. Posttranscriptional gene suppression is mediated by transcription of integrated recombinant DNA to form double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. This formation of dsRNA most commonly results from transcription of an integrated inverted repeat of an element of a target gene, and is a common feature of gene suppression methods known as anti-sense suppression, co-suppression and RNA interference (RNAi). Transcriptional suppression can be mediated by a transcribed dsRNA having homology to a promoter DNA sequence to effect what is called promoter trans suppression.

More particularly, posttranscriptional gene suppression by inserting a recombinant DNA construct with one or more copies of anti-sense oriented DNA to regulate gene expression in plant cells is disclosed in U.S. Pat. No. 5,107,065 (Shewmaker, et al.,) and U.S. Pat. No. 5,759,829 (Shewmaker, et al.,). Transgenic plants transformed using such anti-sense oriented DNA constructs for gene suppression can comprise integrated DNA arranged as an inverted repeats that result from insertion of the DNA construct into plants by Agrobacterium-mediated transformation, as disclosed by Redenbaugh, et al., in “Safety Assessment of Genetically Engineered Flavr Savr™ Tomato, CRC Press, Inc. (1992). Inverted repeat insertions can comprises a part or all of the T-DNA construct, e.g., an inverted repeat of a complete transcription unit or an inverted repeat of transcription terminator sequence. Screening for inserted DNA comprising inverted repeat elements can improve the efficiency of identifying transformation events effective for gene silencing whether the transformation construct is a simple anti-sense DNA construct which must be inserted in multiple copies or a complex inverted repeat DNA construct (e.g., an RNAi construct) which can be inserted as a single copy.

Posttranscriptional gene suppression by inserting a recombinant DNA construct with sense-oriented DNA to regulate gene expression in plants is disclosed in U.S. Pat. No. 5,283,184 (Jorgensen, et al.) and U.S. Pat. No. 5,231,020 (Jorgensen, et al.). Inserted T-DNA providing gene suppression in plants transformed with such sense constructs by Agrobacterium is organized predominately in inverted repeat structures, as disclosed by Jorgensen, et al., Mol. Gen. Genet., 207:471-477 (1987). See also Stam, et al., The Plant Journal, 12(1), 63-82 (1997) who used segregation studies to support Jorgensen's finding that gene silencing is mediated by multimeric transgene T-DNA loci in which the T-DNAs are arranged in inverted repeats. Screening for inserted DNA comprising inverted repeat elements can improve the gene silencing efficiency when transforming with simple sense-orientated DNA constructs. Gene silencing efficiency can also be improved by screening for single insertion events when transforming with an RNAi construct containing inverted repeat elements

As disclosed by Redenbaugh, et al., gene suppression can be achieved by inserting into a plant genome recombinant DNA that transcribes dsRNA. Such a DNA insert can be transcribed to an RNA element having the 3′ region as a double stranded RNA. RNAi constructs are also disclosed in EP 0426195 A1 (Goldbach, et al.,—1991) where recombinant DNA constructs for transcription into hairpin dsRNA for providing transgenic plants with resistance to tobacco spotted wilt virus. Double-stranded RNAs were also disclosed in WO 94/01550 (Agrawal, et al.,) where anti-sense RNA was stabilized with a self-complementary 3′ segment. Agrawal, et al., referred to U.S. Pat. No. 5,107,065 for using such self-stabilized anti-sense RNAs for regulating gene expression in plant cells; see International Publication No. 94/01550. Other double-stranded hairpin-forming elements in transcribed RNA are disclosed in International Publication No. 98/05770 (Werner, et al.,) where the anti-sense RNA is stabilized by hairpin forming repeats of poly(CG) nucleotides. See also U.S. Patent Application Publication No. 2003/0175965 A1 (Lowe, et al.,) which discloses gene suppression using and RNAi construct comprising a gene coding sequence preceded by inverted repeats of 5′UTR. See also U.S. Patent Application Publication No. 2002/0048814 A1 (Oeller) where RNAi constructs are transcribed to sense or anti-sense RNA which is stabilized by a poly(T)-poly(A) tail. See also U.S. Patent Application Publication No. 2003/0018993 A1 (Gutterson, et al.,) where sense or anti-sense RNA is stabilized by an inverted repeat of the 3′ untranslated region of the NOS gene. See also U.S. Patent Application Publication No. 2003/0036197 A1 (Glassman, et al.,) where RNA having homology to a target is stabilized by two complementary RNA regions.

Gene silencing can also be affected by transcribing RNA from both a sense and an anti-sense oriented DNA, e.g., as disclosed by Shewmaker, et al., in U.S. Pat. No. 5,107,065 where in Example 1 a binary vector was prepared with both sense and anti-sense aroA genes. See also U.S. Pat. No. 6,326,193 where gene targeted DNA is operably linked to opposing promoters.

Gene silencing can also be affected by transcribing from contiguous sense and anti-sense DNA. In this regard see Sijen, et al., The Plant Cell, Vol. 8, 2277-2294 (1996) discloses the use of constructs carrying inverted repeats of a cowpea mosaic virus gene in transgenic plants to mediate virus resistance. Such constructs for posttranscriptional gene suppression in plants by double-stranded RNA are also disclosed in International Publication No. WO 99/53050 (Waterhouse, et al.,), International Publication No. WO 99/49029 (Graham, et al.), U.S. 2004-0029283 A1 (Fillatti), U.S. Pat. No. 6,506,559 (Fire, et al.,). See also U.S. 2004-0006792 A1 (Shewmaker, et al.,) that discloses constructs and methods for simultaneously expressing one or more recombinant genes while simultaneously suppressing one or more native genes in a transgenic plant. See also U.S. Pat. No. 6,448,473 (Mitsky, et al.,) that discloses multi-gene suppression vectors for use in plants. All of the above-described patents, applications and international publications disclosing materials and methods for posttranscriptional gene suppression in plants are incorporated herein by reference. Transcriptional suppression such as promoter trans suppression can be affected by a expressing a DNA construct comprising a promoter operably linked to inverted repeats of promoter DNA for a target gene. Constructs useful for such gene suppression mediated by promoter trans suppression are disclosed by Mette, et at, The EMBO Journal, Vol. 18, No. 1, pp. 241-148, 1999 and by Mette, et al., The EMBO Journal, Vol. 19, No. 19, pp. 5194-5201-148, 2000, both of which are incorporated herein by reference.

Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformations with the T-DNA of Agrobacterium are readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that are efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest are identified using the polynucleotides of this invention. For example, a large population of mutated plants are screened to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.

Gene Stacking

This invention also contemplates that the trait-improving recombinant DNA is used in combination with other recombinant DNA to create plants with a multiple desired traits. The combinations generated include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations are created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.

Plant Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA are known in the art and are useful in producing the transgenic seeds of this invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn); and 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.

In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it is useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” means any of the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant is regenerated is useful as a recipient cell. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. 2004-0216189 A1, which are incorporated herein by reference.

In practice DNA is introduced into only a small percentage of target cells in any one experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells are tested further to confirm stable integration of the exogenous DNA. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants are also often employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.

Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in regeneration media and allowed to mature into plants. Developing plantlets are transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28 degrees C. After regenerating plants have reached the stage of shoot and root development, they are transferred to a greenhouse for further growth and testing. Plants are pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.

Progeny are recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.

Discovery of Trait-Improving Recombinant DNA

To identify recombinant DNA that confer improved traits to plants, Arabidopsis plants were transformed with a large population of recombinant DNA constructs for expressing a large variety of distinct DNA. Transgenic plants were produced and screened to identify those plants having recombinant DNA constructs expressing trait-imparting DNA. A two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 2 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.

In particular, Table 2 reports

“PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing. “construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1. “annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit. “description” refers to the description of the top BLAST hit. “e-value” provides the expectation value for the BLAST hit. “identity” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank. “traits” identified by two letters codes the confirmed improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are: “CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen; “CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen; “DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen; “PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen; “HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen; “SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen; “LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen; “LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition; “PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen; “SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.

TABLE 2 Pep SEQ construct annotation Id id gene e value % identity ncbi id description traits 270 14324 CGPG1560 1.00E−127 86 gi|15232185| ref|NP_191546.1|expressed CK SS protein [Arabidopsis thaliana]] 271 17484 CGPG2630 0 93 gi|15220912| ref|NP_173239.1|zinc finger CK (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 272 19109 CGPG1381 9.00E−31 81 gi|18404521| ref|NP_565870.1|expressed CK protein [Arabidopsis thaliana] 273 70423 CGPG3165 1.00E−134 96 gi|30688808| ref|NP_850953.1|MADS- CK CS CK HS PP box protein (AGL9) [Arabidopsis thaliana] gb|AAM65812.1| putative floral homeotic protein, AGL9 [Arabidopsis thaliana] 274 70424 CGPG3180 1.00E−142 81 gi|25405039| pir||H96827protein CK F20B17.12 [imported] - Arabidopsis thaliana gb|AAF68121.1| F20B17.12 [Arabidopsis thaliana] 275 70480 CGPG3833 1.00E−122 99 gi|18411867| ref|NP_565174.1|14-3-3 CK protein GF14 pi (GRF13) [Arabidopsis thaliana] 276 70509 CGPG2420 1.00E−113 82 gi|15225186| ref|NP_180770.1|ovate CK protein-related [Arabidopsis thaliana] 277 70647 CGPG4334 1.00E−171 94 gi|15237269| ref|NP_200093.1|ornithine CK cyclodeaminase/mu- crystallin family protein [Arabidopsis thaliana] dbj|BAB10429.1| 278 70675 CGPG4519 0 100 gi|15224730| ref|NP_180115.1|2- CK oxoglularate-dependent dioxygenase, putative [Arabidopsis thaliana] pir||E84648 probable dioxygenase] 279 70829 CGPG518 0 92 gi|15232841| ref|NP_186854.1|potassium CK transporter (KUP3) [Arabidopsis thaliana] 280 70849 CGPG596 1.00E−166 96 gi|15224801| ref|NP_179547.1|cytidine CK deaminase (CDD)/cytidine aminohydrolase [Arabidopsis thaliana] 281 71627 CGPG1270 0 99 gi|18398032| ref|NP_566315.1|ABC1 CK family protein [Arabidopsis thaliana] 282 71934 CGPG2294 1.00E−154 79 gi|15233973| ref|NP_195575.1|26S CK proteasome regulatory subunit S5A (RPN10) [Arabidopsis thaliana] sp|P55034|PSD4_ARATH 26S proteasome non- ATPase regulatory subunit 4 (26S proteasome regulatory 283 72615 CGPG4829 2.00E−49 88 gi|18422886| ref|NP_568693.1|expressed CK protein [Arabidopsis thaliana] 284 72927 CGPG1477 1.00E−114 81 gi|15234815| ref|NP_194797.1|MA3 CK domain-containing protein [Arabidopsis thaliana] pir||A85359 translation initiation factor-like protein 285 73014 CGPG5692 1.00E−180 93 gi|37528369| ref|NP_931714.1|Fructose- CK PP 1,6-bisphosphatase (D- fructose-1,6-bisphosphate 1-phosphohydrolase) (FBPase) [Photorhabdus luminescens subsp. laumondii TTO1] 286 73559 CGPG6535 0 93 gi|16078422| ref|NP_389241.1|similar to CK aspartate aminotransferase [Bacillus subtilis] sp|O31665|MTNE_BACSU Transaminase mtnE pir||F69863 probable transaminase (EC 2.6.1.-) ykrV 287 74251 CGPG5489 1.00E−171 87 gi|15238437| ref|NP_200760.1|zinc CK SP transporter (ZIP2) [Arabidopsis thaliana] sp|Q9LTH9|ZIP2_ARATH Zinc transporter 2 precursor (ZRT/IRT-like protein 2) 288 19631 CGPG3627 1.00E−94 90 gi|18410249| ref|NP_565053.1|SNF7 CS family protein [Arabidopsis thaliana] pir||G96755 developmental protein homolog DG1118 [imported] - Arabidopsis thaliana 289 70121 CGPG2380 1.00E−111 100 gi|6323765| ref|NP_013836.1|Hypothetical CS ORF; Ymr118cp [Saccharomyces cerevisiae] sp|Q04487|YM07_YEAST Putative succinate dehydrogenase cytochrome B subunit, mitochondrial precursor 290 70654 CGPG4352 2.00E−63 88 gi|18400941| ref|NP_566531.1|expressed CS protein [Arabidopsis thaliana] 291 70696 CGPG4590 8.00E−92 87 gi|25408379| pir||E84768hypothetical CS PP protein At2g35430 292 70713 CGPG1462 0 95 gi|30678679| ref|NP_191966.2|malate CS oxidoreductase, putative 293 70740 CGPG3700 0 94 gi|18402759| ref|NP_566667.1|transcription CS LL PP factor jumonji (jmjC) domain-containing protein 294 71321 CGPG4418 0 91 gi|13878402| sp|Q9STL0|C71N_ARATH CS Cytochrome P450 71A23 pir||T06712 probable cytochrome P450 T29H11.180 295 71835 CGPG4634 0 100 gi|15234361| ref|NP_192100.1|DC1 CS SP domain-containing protein [Arabidopsis thaliana] pir||E85024 probable CHP- rich zinc finger protein 296 72934 CGPG5798 0 98 gi|6323512| ref|NP_013583.1|High- CS affinity inorganic phosphate (Pi) transporter and low- affinity manganese transporter; regulated by Pho4p and Spt7p; mutation confers resistance to arsenate; exit from the ER during maturation requires Pho86p; Pho84p [Saccharomyces cerevisiae] 297 72945 CGPG5787 0 94 gi|6319991| ref|NP_010071.1|GABA- CS specific transport protein; Uga4p [Saccharomyces cerevisiae] sp|P32837|UGA4_YEAST GABA-specific permease (GABA-specific transport protein) 298 72980 CGPG5773 0 89 gi|6321960| ref|NP_012036.1|Subunit of CS the anaphase-promoting complex/cyclosome (APC/C), which is a ubiquitin-protein ligase required for degradation of anaphase inhibitors, including mitotic cyclins, during the metaphase/anaphase transition; Cdc23p 299 73504 CGPG6480 1.00E−178 100 gi|16330153| ref|NP_440881.1|fructokinase CS PP [Synechocystis sp. PCC 6803] pir||S77227 fructokinase (EC 2.7.1.4) - Synechocystis sp. (strain PCC 6803) 300 73507 CGPG6504 1.00E−173 100 gi|16078547| ref|NP_389366.1|similar to CS LL PP PEG glutaminase [Bacillus subtilis] 301 73573 CGPG6462 0 100 gi|15890038| ref|NP_355719.1|AGR_C_ CS PP 5067p [Agrobacterium tumefaciens str. C58] ref|NP_533456.1| 3- isopropylmalate dehydrogenase 302 73586 CGPG6471 1.00E−77 100 gi|16077684| ref|NP_388498.1|similar to CS DS PP fructokinase [Bacillus subtilis] 303 73770 CGPG5435 2.00E−34 68 gi|15235771| ref|NP_193383.1|cysteine CS PP protease inhibitor family protein/cystatin family protein [Arabidopsis thaliana] 304 74105 CGPG6574 0 73 gi|23059330| ref|ZP_00084307.1|COG10 CS 12: NAD-dependent aldehyde dehydrogenases [Pseudomonas fluorescens PfO-1] 305 74111 CGPG6622 0 99 gi|16131442| ref|NP_418028.1|alpha- CS amylase [Escherichia coli K12] 306 74136 CGPG6632 9.00E−81 99 gi|16330993| ref|NP_441721.1|unknown CS CK HS protein [Synechocystis sp. PCC 6803] 307 74139 CGPG6561 1.00E−180 95 gi|24112825| ref|NP_707335.1|glyceralde CS LL hyde-3-phosphate dehydrogenase A [Shigella flexneri 2a str. 301] 308 74267 CGPG5364 0 97 gi|18399375| ref|NP_566402.1|U-box CS domain-containing protein [Arabidopsis thaliana] 309 74291 CGPG5363 0 94 gi|18401867| ref|NP_565676.1|armadillo/ CS beta-catenin repeat family protein/U-box domain- containing protein [Arabidopsis thaliana] 310 74318 CGPG5826 0 100 gi|15219730| ref|NP_176847.1|cell CS HS division protein kinase, putative [Arabidopsis thaliana] 311 74319 CGPG5831 0 96 gi|15224359| ref|NP_181907.1|mitogen- CS activated protein kinase, putative/MAPK, putative (MPK6) [Arabidopsis thaliana] 312 74324 CGPG5885 1.00E−174 95 gi|42569304| ref|NP_180094.2|protein CS kinase family protein [Arabidopsis thaliana] 313 74512 CGPG32 0 96 gi|15217945| ref|NP_176132.1|amino CS HS SP acid permease I (AAP1) [Arabidopsis thaliana] 314 74583 CGPG6649 1.00E−151 83 gi|22978283| ref|ZP_00024043.1|COG02 CS PP 52: L- asparaginase/archaeal Glu- tRNAGln amidotransferase subunit D [Ralstonia metallidurans] 315 70427 CGPG3067 0 100 gi|42572771| ref|NP_974481.1|kelch CS DS repeat-containing F-box family protein [Arabidopsis thaliana] 316 71811 CGPG4426 0 97 gi|15223341| ref|NP_171627.1|cytochrome CS DS LL LN P450,putative [Arabidopsis thaliana] 317 73463 CGPG6384 0 100 gi|22977164| ref|ZP_00022985.1|COG05 DS 38: Isocitrate dehydrogenases [Ralstonia metallidurans] 318 72081 CGPG5279 7.00E−66 77 gi|42570373| ref|NP_850277.2|CCAAT- DS PEG box binding transcription factor, putative [Arabidopsis thaliana] 319 10139 CGPG101 0 89 gi|15229877| ref|NP_187154.1|sodium DS proton exchanger, putative (NHX2) [Arabidopsis thaliana] 320 11410 CGPG103 0 82 gi|15236418| ref|NP_192555.1|homeobox DS protein knotted-1 like 1 (KNAT1)[Arabidopsis thaliana] 321 11604 CGPG48 0 92 gi|15233457| ref|NP_194642.1|hexokinase DS 1 (HXK1) [Arabidopsis thaliana] 322 12368 CGPG1006 1.00E−146 85 gi|15231451| ref|NP_190238.1|epsin N- DS terminal homology (ENTH) domain-containing protein/ clathrin assembly protein- related [Arabidopsis thaliana] 323 13502 CGPG1354 0 95 gi|15224557| ref|NP_180632.1|serine/ DS PP threonine protein kinase, putative [Arabidopsis thaliana] 324 13745 CGPG1576 1.00E−112 84 gi|15222987| ref|NP_177749.1|hypothetical DS protein [Arabidopsis thaliana] gb|AAF17642.1| T23E18.15 [Arabidopsis thaliana] 325 13821 CGPG1569 1.00E−155 85 gi|18416499| ref|NP_567716.1|expressed DS protein [Arabidopsis thaliana) 326 14240 CGPG1697 0 94 gi|15241302| ref|NP_197527.1|expressed DS protein [Arabidopsis thaliana] 327 14718 CGPG1082 0 86 gi|18407200| ref|NP_566090.1|expressed DS protein [Arabidopsis thaliana] 328 17022 CGPG1774 1.00E−159 100 gi|15237803| ref|NP_197755.1|nodulin DS MtN3 Family protein [Arabidopsis thaliana] 329 17924 CGPG2882 3.00E−92 100 gi|15233350| ref|NP_192875.1|zinc finger DS (C3HC-4-type RING finger) family protein (RHA1b) [Arabidopsis thaliana] 330 18259 CGPG3368 2.00E−94 88 gi|30685085| ref|NP_849549.1|zinc finger DS PP protein (LSD1) [Arabidopsis thaliana] 331 19171 CGPG2952 0 91 gi|6320063| ref|NP_010143.1|plasma DS membrane glucose sensor; Rgt2p [Saccharomyces cerevisiae] 332 19201 CGPG2332 0 96 gi|15233948| ref|NP_194205.1|protein DS kinase (AFC2) [Arabidopsis thaliana] sp|P51567|AFC2_ARATH Protein kinase 333 19317 CGPG3662 1.00E−151 91 gi|21232858| ref|NP_638775.1|conserved DS hypothetical protein [Xanthomonas campestris pv. campestris str. ATCC 33913] 334 70417 CGPG3427 0 81 gi|18396278| ref|NP_566180.1|integral DS PP SP membrane family protein [Arabidopsis thaliana] 335 70467 CGPG3785 0 100 gi|15241416| ref|NP_196953.1|no apical DS meristem (NAM) family protein [Arabidopsis thaliana] 336 70806 CGPG712 0 100 gi|15218225| ref|NP_173010.1|cyclin, DS putative [Arabidopsis thaliana] 337 70818 CGPG479 1.00E−157 92 gi|30691978| ref|NP_568508.2|bZIP DS transcription factor family protein [Arabidopsis thaliana] 338 70820 CGPG655 0 93 gi|15224342| ref|NP_181899.1|acyl-[acyl- DS carrier-protein] desaturase/ stearoyl-ACP desaturase (SSI2) [Arabidopsis thaliana] 339 70919 CGPG4029 1.00E−169 73 gi|6996560| emb|CAB75429.1|oligouridy- DS late binding protein [Nicotiana plumbaginifolia] 340 71623 CGPG4696 1.00E−150 95 gi|15236511| ref|NP_192588.1|mitogen- DS activated protein kinase, putative [Arabidopsis thaliana] pir||T01835 serine/threonine-specific protein kinase ARA.KIN homolog T15F16.3 - Arabidopsis thaliana 341 71662 CGPG4679 1.00E−173 91 gi|5929964| gb|AAD56659.1|malate DS dehydrogenase [Glycine max] 342 71693 CGPG4652 6.00E−86 56 gi|21553460| gb|AAM62553.1|snap25a DS [Arabidopsis thaliana] 343 72384 CGPG4639 0 98 gi|1169548| sp|P38604|ERG7_YEASTL DS anosterol synthase (Oxidosqualene--lanosterol cyclase) (2,3- epoxysqualene--lanosterol cyclase) (OSC) gb|AAA64377.1|2,3- oxidosqualene-lanosterol cyclase 344 72439 CGPG5075 8.00E−57 90 gi|22331337| ref|NP_683594.1|NPR1/NI DS M1-interacting protein 2 (NIMIN-2) 345 72619 CGPG4835 6.00E−83 88 gi|15237317| ref|NP_200108.1|expressed DS protein [Arabidopsis thaliana] 346 72624 CGPG4842 0 100 gi|15242148| ref|NP200558.1|expressed DS protein [Arabidopsis thaliana] 347 72715 CGPG5521 0 91 gi|6323933| ref|NP_014004.1|Carboxy- DS SS terminal domain (CTD) phosphatase, essential for dephosphorylation of the repeated C-terminal domain of the RNA polymerase II large subunit (Rpo21p); Fcp1p [Saccharomyces cerevisiae] 348 72754 CGPG5548 1.00E−169 100 gi|728961| sp|Q00618|BET4_YEASTG DS eranylgeranyl transferase type II alpha subunit (Type II protein geranyl- 349 72819 CGPG4989 0 100 gi|18417026| ref|NP_567780.1|pfkB-type DS carbohydrate kinase family protein [Arabidopsis thaliana] 350 75516 CGPG7689 1.00E−138 70 gi|42568081| ref|NP_197938.2|zinc finger DS (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 351 75701 CGPG7856 8.00E−41 37 gi|15225413| ref|NP_182037.1|zinc finger DS LN (C2H2 type) family protein [Arabidopsis thaliana] 352 73515 CGPG6473 1.00E−162 100 gi|16079626| ref|NP_390450.1|similar to HS CS PEG 6-phosphogluconate dehydrogenase (pentose phosphate) [Bacillus subtilis] 353 74684 CGPG6360 1.00E−64 100 gi|18390735| ref|NP_563782.1|expressed CS HS protein [Arabidopsis thaliana] 354 19542 CGPG3069 0 90 gi|18403574| ref|NP_564592.1|F-box HS family protein [Arabidopsis thaliana] 355 19618 CGPG3574 1.00E−121 100 gi|15218423| ref|NP_177373.1|trypsin HS and protease inhibitor family protein/Kunitz family protein [Arabidopsis thaliana] pir||F96746 probable drought induced protein 356 19649 CGPG3140 1.00E−141 87 gi|18412787| ref|NP_567287.1|vesicle- HS associated membrane family protein/VAMP family protein 357 19745 CGPG3973 2.00E−60 46 gi|15239303| ref|NP_201424.1|expressed HS protein [Arabidopsis thaliana] 358 19768 CGPG4096 1.00E−179 81 gi|25052804| gb|AAN65180.1|mitogen- HS SS activated protein kinase 4 [Petroselinum crispurn] 359 19772 CGPG3939 3.00E−82 79 gi|7488744| pir||T09700MADS-box HS protein-alfalfa (fragment) gb|AAB51377.1|MADS-box protein [Medicago sativa] 360 19779 CGPG4113 1.00E−153 89 gi|30681126| ref|NP_196201.2|phosphate CS HS translocator-related [Arabidopsis thaliana] 361 19833 CGPG4074 1.00E−107 79 gi|6683777| gb|AAF23363.1|CAGL2 CS HS PP [Cucumis sativus] 362 19862 CGPG3961 2.00E−89 56 gi|15229637| ref|NP_188469.1|no apical HS meristem (NAM) family protein [Arabidopsis thaliana] dbj|BAB01106.11 unnamed protein product [Arabidopsis thaliana] 363 19879 CGPG4009 0 75 gi|18401703| ref|NP_564504.1|protein HS CS SS phosphatase 2C-related/ PP2C-related [Arabidopsis thaliana] 364 70445 CGPG3728 2.00E−51 88 gi|30696602| ref|NP_200357.2|protease HS inhibitor/seed storage/lipid transfer protein (LTP) family protein [Arabidopsis thaliana] 365 70738 CGPG3195 1.00E−96 100 gi|15234797| ref|NP_194791.1|expressed HS PP protein [Arabidopsis thaliana] 366 71437 CGPG4043 1.00E−164 81 gi|15241535| ref|NP_196433.1|serine/ HS threonine-protein kinase, putative [Arabidopsis thaliana] 367 71572 CGPG4520 3.00E−83 92 gi|18403850| ref|NP_565804.1|expressed HS protein [Arabidopsis thaliana] 368 71617 CGPG1227 0 100 gi|15236219| ref|NP_195218.1|1- HS CK phosphatidylinositol phosphodiesterase-related [Arabidopsis thaliana] 369 72532 CGPG4780 1.00E−118 90 gi|15236659| ref|NP_194120.1|expressed HS protein [Arabidopsis thaliana] 370 72757 CGPG5572 0 89 gi|15242402| ref|NP_197088.1|zinc finger HS LL PEG protein-CONSTANS (CO) [Arabidopsis thaliana] 371 73412 CGPG6448 0 99 gi|28867589| ref|NP_790208.1|glutamine HS synthetase, type I [Pseudomonas syringae pv. tomato str. DC3000] 372 74102 CGPG6550 1.00E−167 94 gi|15614187| ref|NP_242490.1|L- HS asparaginase [Bacillus halodurans C-125] 373 72633 CGPG4853 1.00E−145 86 gi|15238013| ref|NP_199519.1|casein CS LL PEG kinase II beta chain, putative [Arabidopsis thaliana] 374 72456 CGPG4745 1.00E−75 92 gi|15239846| ref|NP_196763.1|17.6 kDa DS LL class II heat shock protein (HSP17.6-CII) [Arabidopsis thaliana] 375 72963 CGPG1746 1.00E−151 87 gi|15222239| ref|NP_172174.1|ovate LL LN family-protein [Arabidopsis thaliana] 376 70426 CGPG3199 8.00E−54 88 gi|18397268| ref|NP_564336.1|double- LL stranded DNA-binding family protein [Arabidopsis thaliana] 377 70772 CGPG4627 1.00E−92 80 gi|15220084| ref|NP_173175.1|MADS- LL box protein (AGL100) [Arabidopsis thaliana] P 378 71137 CGPG125 1.00E−111 90 gi|15218957| ref|NP_176202.1|two- LL component responsive regulator/response regulator 3 (ARR3) [Arabidopsis thaliana] 379 71529 CGPG2808 1.00E−131 72 gi|42562375| ref|NP_174152.3|Dof-type LL zinc finger domain- containing protein [Arabidopsis thaliana] 380 71601 CGPG1858 1.00E−168 92 gi|15231425| ref|NP_187378.1|transcrip- LL tional activator, putative [Arabidopsis thaliana] 381 72362 CGPG983 1.00E−163 95 gi|15242779| ref|NP_200562.1|xyloglucan: LL xyloglucosyl transferase, putative/xyloglucan endotransglycosylase, putative/endo-xyloglucan transferase, putative [Arabidopsis thaliana] 382 72466 CGPG4767 3.00E−60 83 gi|15234046| ref|NP_195030.1|glutaredoxin CK LL PEG family protein [Arabidopsis thaliana] 383 72524 CGPG4770 1.00E−134 91 gi|18412649| ref|NP_567140.1|expressed CK LL protein [Arabidopsis thaliana] 384 73085 CGPG5689 1.00E−134 100 gi|16331347| ref|NP_442075.1| LL triosephosphate isomerase [Synechocystis sp. PCC 6803] 385 74241 CGPG5457 0 89 gi|444790| prf||1908224Anucleotide LL translocator 386 74247 CGPG5475 1.00E−159 100 gi|18411863| ref|NP_565172.1|protein LL phosphatase 2C, putative/ PP2C, putative [Arabidopsis thaliana] 387 74284 CGPG5413 0 97 gi|15230577| ref|NP_190087.1|serine LL carboxypeptidase III, putative [Arabidopsis thaliana] 388 74652 CGPG6168 1.00E−90 80 gi|15235970| ref|NP_194879.1|expressed LL DS protein [Arabidopsis thaliana] 389 70437 CGPG3706 1.00E−172 100 gi|30678824| ref|NP_186983.2|short- CK LN chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] 390 71633 CGPG857 1.00E−100 86 gi|6690274| gb|AAF24061.1|v-SNARE DS LN AtVTI1a [Arabidopsis thaliana] 391 72948 CGPG5617 0 94 gi|15225456| ref|NP_182059.1|leucine- LN PEG rich repeat transmembrane protein kinase, putative [Arabidopsis thaliana] 392 72519 CGPG4749 LN SS 393 10475 CGPG399 1.00E−164 96 gi|15240972| ref|NP_195761.1|stress- LN responsive protein, putative [Arabidopsis thaliana] 394 11120 CGPG459 0 100 gi|15227169| ref|NP_179812.1|inositol-3- LN phosphate synthase isozyme 2/myo-inositol-1- phosphate synthase 2/MI- 1-P synthase 2/IPS 2 [Arabidopsis thaliana] 395 19736 CGPG4129 2.00E−94 67 gi|13346194| gb|AAK19619.1|GHMYB9 LN [Gossypium hirsutum] 396 71606 CGPG4715 0 91 gi|15218674| ref|NP_171800.1|phototropic- LN responsive NPH3 family protein [Arabidopsis thaliana] 397 71840 CGPG4353 0 96 gi|18401087| ref|NP_566542.1|mitotic DS LL LN phosphoprotein N′ end (MPPN) family protein [Arabidopsis thaliana] 398 74240 CGPG5454 1.00E−155 90 gi|15233884| ref|NP_194188.1|mitochondrial CK LN substrate carrier family protein [Arabidopsis thaliana] pir||T05577 uncoupling protein homolog F22K18.230-Arabidopsis thaliana 399 74331 CGPG5834 0 94 gi|15220416| ref|NP_172003.1|protein LN kinase familyprotein [Arabidopsis thaliana] 400 74610 CGPG6048 1.00E−117 100 gi|15217568| ref|NP_172434.1|Ras- LL LN related GTP-binding protein, putative [Arabidopsis thaliana] sp|O04486|RB1C_ARATH Ras-related protein Rab11C 401 75527 CGPG7682 1.00E−55 65 gi|15240946| ref|NP_195750.1|phosphatidyl- LN ethanolamine-binding family protein [Arabidopsis thaliana] 402 70681 CGPG4584 9.00E−64 93 gi|18411465| ref|NP_567196.1|auxin- CK PEG responsive family protein [Arabidopsis thaliana] 403 71663 CGPG4638 0 93 gi|21230153| ref|NP_636070.1|conserved CK PEG hypothetical protein [Xanthomonas campestris pv. campestris str. ATCC 33913] 404 72769 CGPG5573 0 100 gi|15225499| ref|NP_182075.1|cytochrome CK PEG P450, putative [Arabidopsis thaliana] 405 71508 CGPG1541 2.00E−24 100 gi|15241504| sp|Q9SD80|OM05_ARATH PEG CS SP PEG Mitochondrial import receptor subunit TOM5 homolog (Translocase of outer membrane 5 kDa subunit homolog) 406 74248 CGPG5476 0 100 gi|15226152| ref|NP_180926.1|protein PEG CS phosphatase 2C, putative/ PP2C, putative [Arabidopsis thaliana] p 407 72771 CGPG2166 5.00E−44 100 gi|18395032| ref|NP_564151.1|expressed PEG CK HS SS protein [Arabidopsis thaliana] 408 72085 CGPG5228 0 94 gi|15241541| ref|NP_199275.1|cytochrome PEG HS P450 family protein [Arabidopsis thaliana] dbj|BAA98115.1|flavonoid 3′,5′-hydroxylase-like; cytochrome P450 [Arabidopsis thaliana] 409 72744 CGPG5563 1.00E−136 96 gi|6321574| ref|NP_011651.1|20S HS PEG CK proteasome beta-type subunit; the only nonessential 20S subunit; Pre9p [Saccharomyces cerevisiae] 410 73039 CGPG810 0 96 gi|15242124| ref|NP_197599.1|molybdop- HS PEG terin biosynthesis CNX1 protein/molybdenum cofactor biosynthesis enzyme CNX1 (CNX1) [Arabidopsis thaliana] 411 73054 CGPG5754 2.00E−98 100 gi|6324827| ref|NP_014896.1|Nat5p PEG HS SS PEG [Saccharomyces cerevisiae] pir||S67150 hypothetical protein YOR253w - yeast (Saccharomyces cerevisiae) 412 73501 CGPG6456 0 97 gi|15888752| ref|NP_354433.1|AGR_C_ HS PEG 2631p [Agrobacterium tumefaciens str. C58] sp|Q8UFH1|ENO_AGRT5 Enolase (2- phosphoglycerate dehydratase) (2-phospho- D-glycerate hydro-lyase) 413 19707 CGPG4179 1.00E−86 49 gi|15236282| ref|NP_195242.1|O- PEG CS methyltransferase family 2 protein [Arabidopsis thaliana] 414 19951 CGPG3941 5.00E−91 54 gi|15221582| ref|NP_177064.1|basic PEG CK helix-loop-helix (bHLH) family protein [Arabidopsis thaliana] 415 19967 CGPG4032 1.00E−127 67 gi|4760710| dbj|BAA77395.1|SLL2-S9- PEG protein [Brassica rapa] 416 70543 CGPG3815 0 95 gi|15220994| ref|NP_175222.1|E2F PEG CK transcription factor-2 (E2F2)/transcription factor E2Fc (E2Fc) [Arabidopsis thaliana] 417 70707 CGPG1273 1.00E−108 100 gi|15219558| ref|NP_177523.1|Ssu72- PEG like family protein [Arabidopsis thaliana] pir||F96765 unknown protein F 418 70719 CGPG1712 0 86 gi|18394560| ref|NP_564043.1|expressed PEG protein [Arabidopsis thaliana] 419 71134 CGPG817 8.00E−55 100 gi|15240471| ref|NP_200327.1|small PEG HS PP ubiquitin-like modifier 2 (SUMO) [Arabidopsis thaliana] 420 71146 CGPG2928 1.00E−86 92 gi|29165403| gb|AAO65311.1|MADS PEG affecting flowering 3 variant II [Arabidopsis thaliana] 421 71660 CGPG4690 3.00E−80 100 gi|18415773| ref|NP_567637.1|methionine PEG sulfoxide reductase domain-containing protein/ SelR domain-containing protein [Arabidopsis thaliana] 422 72086 CGPG5236 0 100 gi|15232215| ref|NP_191556.1|methyl- PEG PP PEG enetetrahydrofolate reductase 1 (MTHFR1) [Arabidopsis thaliana]- 423 72632 CGPG4852 4.00E−99 90 gi|18425032| ref|NP_569028.1|expressed PEG protein [Arabidopsis thaliana] 424 72716 CGPG5529 2.00E−85 89 gi|6320196| ref|NP_010276.1|subunit of PEG the Anaphase Promoting Complex; all known APC subunits co- immunoprecipitate with epitope-tagged Apc11p; Apc11p [Saccharomyces cerevisiae] 425 72723 CGPG1848 0 97 gi|15237500| ref|NP_199487.1|human PEG Rev interacting-like family protein/hRIP family protein [Arabidopsis thaliana] dbj|BAB08919.1|zinc finger protein Glo3-like [Arabidopsis thaliana] 426 72987 CGPG1787 5.00E−81 77 gi|15231568| ref|NP_189282.1|octicosa- PEG SP peptide/Phox/Bem1p (PB1) domain-containing protein [Arabidopsis thaliana] 427 74109 CGPG6606 0 77 gi|37524479| ref|NP_927823.1|malto- PEG dextrin phosphorylase [Photorhabdus luminescens subsp. laumondii TTO1] 428 74140 CGPG6569 0 99 gi|15613102| ref|NP_241405.1|NADP- PEG PP PEG dependent aldehyde dehydrogenase [Bacillus halodurans C-125] 429 74191 CGPG6597 0 96 gi|22960294| ref|ZP_00007935.1|COG18 PEG 50: Ribulose 1.5- bisphosphate carboxylase, large subunit [Rhodobacter sphaeroides] 430 74265 CGPG5356 1.00E−117 100 gi|15237288| ref|NP_197727.1|GRAM PEG PP PEG domain-containing protein/ ABA-responsive protein- related [Arabidopsis thaliana] 431 74369 CGPG6076 2.00E−86 96 gi|18409647| ref|NP_564994.1|ubiquitin- PEG CK PP PEG conjugating enzyme family protein [Arabidopsis thaliana] 432 70217 CGPG6 0 97 gi|15231536| ref|NP189259.1|cytochrome CK PP SP P450 family protein [Arabidopsis thaliana] 433 72711 CGPG1846 4.00E−75 79 gi|15221048| ref|NP_175816.1|transcription CK PP SP initiation factor IID (TFIID) 31 kDa subunit (TAFII-31) family protein [Arabidopsis thaliana] 434 70932 CGPG4089 1.00E−129 56 gi|15223134| ref|NP_177792.1|expressed CS HS PP protein [Arabidopsis thaliana] 435 73518 CGPG6497 1.00E−177 63 gi|22981996| ref|ZP_00027327.1|COG10 CS CK PP 12: NAD-dependent aldehyde dehydrogenases [Burkholderia fungorum] 436 19771 CGPG4011 3.00E−90 80 gi|18418200| ref|NP_568342.1|rubredoxin PP HS SS family protein [Arabidopsis thaliana] dbj|BAB10504.1| gene_id:MKP11.2~unknown protein [Arabidopsis thaliana] g 437 73549 CGPG6460 0 90 gi|37524978| ref|NP_928322.1|5- HS DS PP carboxymethyl-2- hydroxymuconate semialdehyde dehydrogenase [Photorhabdus luminescens subsp. laumondii TTO1] 438 72994 CGPG5803 0 83 gi|6322702| ref|NP_012776.1|Vacuolar CK PEG CS PP PEG transporter, exports large neutral amino acids from the vacuole; member of a family of seven S. cerevisiae genes (AVT1-7) related to vesicular GABA- glycine transporters; Avt3p [Saccharomyces cerevisiae] 439 71928 CGPG1617 0 100 gi|18394888| ref|NP_564120.1|catalase 3 CS PEG CK PP PEG (SEN2) [Arabidopsis thaliana] 440 72903 CGPG5584 0 91 gi|6322293| ref|NP_012367.1|Histone PEG PP SS methyltransferase with a role in transcriptional elongation, methylates a lysine residue of histone H3; associates with the C- terminal domain of Rpo21p; histone methylation activity is regulated by phosphorylation status of Rpo21p; Set2p [Saccharomyces cerevisiae] sp|P46995|SET2_YEAST SET domain protein 2 441 73017 CGPG5733 0 94 gi|6325368| ref|NP_015436.1|kinase PEG PP PEG required for late nuclear division; Dbf20p [Saccharomyces cerevisiae] 442 74587 CGPG6774 0 94 gi|17938451| ref|NP_535240.1|succinate PEG DS HS PP SS semialdehyde dehydrogenase [Agrobacterium tumefaciens str. C58] 443 72453 CGPG4735 6.00E−67 91 gi|15218924| ref|NP_174236.1|auxin- CK PP SP SS responsive family protein [Arabidopsis thaliana] pir||A86417 probable auxin- induced protein, 45653- 45228 444 72967 CGPG5742 0 99 gi|6321525| ref|NP_011602.1|Cytosolic CS CK HS LL PP SS catalase T, has a role in protection from oxidative damage by hydrogen peroxide; Ctt1p [Saccharomyces cerevisiae] 445 72961 CGPG5591 0 95 gi|15228498| ref|NP_186975.1|UTP-- PEG SS HS PP glucose-1-phosphate uridylyltransferase, putative/ UDP-glucose pyrophosphorylase, putative/UGPase, putative [Arabidopsis thaliana] 446 73070 CGPG5627 0 90 gi|15225044| ref|NP_181451.1|protein PEG PP SS kinase family protein [Arabidopsis thaliana] 447 73475 CGPG6385 0 100 gi|39934021| ref|NP_946297.1|glyceralde- PEG PP SS hyde-3-phosphate dehydrogenase(GAPDH) [Rhodopseudomonas palustris CGA009] 448 72916 CGPG1814 0 97 gi|15228871| ref|NP_188303.1|protein PP SS phosphatase 2C, putative/ PP2C, putative [Arabidopsis thaliana] 449 72969 CGPG5789 0 94 gi|6321886| ref|NP_011962.1|Low- PP SP SS affinity glucose transporter of the major facilitator superfamily, expression is induced by Hxk2p in the presence of glucose and repressed by Rgt1p when glucose is limiting; Hxt1p [Saccharomyces cerevisiae] 450 74449 CGPG6659 0 96 gi|15890426| ref|NP_356098.1|AGR_L_ PP SS 619p [Agrobacterium tumefaciens str. C58] pir||A98170 hypothetical protein AGR_L_619 [imported]-Agrobacterium 451 16615 CGPG2539 0 98 gi|15890896| ref|NP_356568.1|AGR_L_ PP 1560p [Agrobacterium tumefaciens str. C58] ref|NP_534561.1| glucose- 1-phosphate adenylyltransferase [Agrobacterium tumefaciens str. C58] 452 19187 CGPG3310 0 91 gi|18423163| ref|NP_568731.1|squamosa PP promoter-binding protein, putative [Arabidopsis thaliana] 453 19648 CGPG3134 1.00E−179 96 gi|18413950| ref|NP_568102.1|short- PP chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] 454 70354 CGPG3995 0 64 gi|15241312| ref|NP_196916.1|nodulin DS PP SP family protein [Arabidopsis thaliana] 455 70421 CGPG2942 0 88 gi|30677977| ref|NP_178317.2|zinc finger PP (C2H2 type) family protein [Arabidopsis thaliana] 456 70459 CGPG3758 0 95 gi|15233315| ref|NP_188242.1|F-box PP family protein[Arabidopsis thaliana] dbj|BAB01261.1| unnamed protein product [Arabidopsis thaliana] 457 70465 CGPG3775 1.00E−155 90 gi|15236937| ref|NP_195254.1|zinc finger PP SP (C2H2 type) family protein [Arabidopsis thaliana] 458 70683 CGPG4587 3.00E−65 64 gi|18423239| ref|NP_568751.1|poly- PP adenylate-binding protein, putative/PABP, putative [Arabidopsis thaliana] 459 70725 CGPG2097 0 91 gi|18420505| ref|NP_568066.1|expressed CS PP protein [Arabidopsis thaliana] 460 70852 CGPG1465 0 93 gi|15237075| ref|NP_195290.1|isocitrate PP SP dehydrogenase, putative/ NAD+ isocitrate dehydrogenase, putative [Arabidopsis thaliana] 461 71112 CGPG934 1.00E−130 94 gi|15218701| ref|NP_171806.1|expressed CS PP protein [Arabidopsis thaliana] pir||E86161 F10O3.11 protein- Arabidopsis thaliana gb|AAD25802.1| Belongs to the PF|01027 Uncharacterized protein family UPF0005 with 7 transmembrane domains. [Arabidopsis thaliana] 462 71127 CGPG945 0 97 gi|15225307| ref|NP_179604.1|26S PP protease regulatory complex subunit 4, putative [Arabidopsis thaliana] pir||E84585 26S proteasome subunit 4 [imported]-Arabidopsis thaliana 463 71132 CGPG1561 0 98 gi|15232209| ref|NP_191550.1|expressed PP protein [Arabidopsis thaliana] 464 71217 CGPG95 0 100 gi|15221476| ref|NP_172127.1|shaggy- PP related protein kinase iota/ ASK-iota (ASK9) (GSK1) [Arabidopsis thaliana] (EC 2.7.1.-) 465 71645 CGPG4688 3.00E−69 100 gi|18401105| ref|NP_566544.1|phospho- PP transfer family protein [Arabidopsis thaliana] 466 71726 CGPG3894 0 93 gi|15217677| ref|NP_171725.1|no apical HS PP meristem (NAM) family protein [Arabidopsis thaliana] 467 72432 CGPG4562 1.00E−144 92 gi|20152540| emb|CAD29662.1|putative PP SP auxin response factor 23 [Arabidopsis thaliana] 468 72450 CGPG4732 1.00E−170 100 gi|15238890| ref|NP_197366.1|zinc finger LL PP (C3HC4-type RING finger) family protein [Arabidopsis thaliana] 469 72455 CGPG4742 1.00E−144 93 gi|15242893| ref|NP_200597.1|anthranilate PP PEG synthase beta subunit, putative [Arabidopsis thaliana] 470 72727 CGPG5522 1.00E−118 100 gi|6324107| ref|NP_014177.1|functionally PP related to TFIIB, affects start site selection in vivo; Ssu72p [Saccharomyces cerevisiae] 471 72817 CGPG4987 0 96 gi|30679158| ref|NP_567238.2|AAA-type PP ATPase family protein [Arabidopsis 472 72992 CGPG5777 0 90 gi|6324981| ref|NP_015049.1|S- PP PEG adenosylMethionine Permease; Sam3p [Saccharomyces cerevisiae] 473 73007 CGPG5760 0 93 gi|6320865| ref|NP_010944.1|One of PP three possible beta- subunits of the Snf1 kinase complex, allows nuclear localization of the Snf1 kinase complex in the presence of a nonfermentable carbon source; contains glycogen- binding domain; Gal83p [Saccharomyces cerevisiae] 474 73073 CGPG5688 0 95 gi|16331010| ref|NP_441738.1|fructose PP 1,6-bisphosphatase [Synechocystis sp. PCC 6803] 475 73506 CGPG6496 0 96 gi|23062569| ref|ZP_00087347.1|COG10 PP 12: NAD-dependent aldehyde dehydrogenases [Pseudomonas fluorescens PfO-1] 476 74107 CGPG6590 0 95 gi|15965198| ref|NP_385551.1|PYRUVATE PP SS DEHYDROGENASE ALPHA2 SUBUNIT PROTEIN [Sinorhizobium meliloti 1021] 477 74117 CGPG6575 0 81 gi|37528116| ref|NP_931461.1|Phenyl- CS PP acetaldehyde dehydrogenase (PAD) [Photorhabdus luminescens subsp. laumondii TTO1] 478 74131 CGPG6592 0 96 gi|16329404| ref|NP_440132.1|trans- PP SS aldolase [Synechocystis sp. PCC 6803] B - 479 74344 CGPG5929 1.00E−111 100 gi|15236410| ref|NP_193147.1|COP9 HS PP signalosome subunit, putative/CSN subunit. putative (CSN8) [Arabidopsis thaliana] 480 14320 CGPG1229 0 100 gi|18418018| ref|NP_567894.1|expressed SP protein [Arabidopsis thaliana] 481 16756 CGPG2117 1.00E−142 85 gi|18391249| ref|NP_563885.1|expressed SP protein [Arabidopsis thaliana] 482 17448 CGPG2673 1.00E−102 72 gi|15239624| ref|NP_197993.1|PHD SP finger Family protein [Arabidopsis thaliana] gb|AAM64729.1| nucleic acid binding protein-like [Arabidopsis thaliana] 483 17633 CGPG2839 1.00E−145 85 gi|18395124| ref|NP_564171.1|basic SP helix-loop-helix (bHLH) family protein [Arabidopsis thaliana] 484 18876 CGPG3096 1.00E−172 89 gi|18394949| ref|NP_564133.1|transporter- SP related [Arabidopsis thaliana] pir||G86343 hypothetical protein T22|11.10 485 19120 CGPG1976 0 100 gi|15232345| ref|NP_188710.1|fertilization- SP independent endosperm protein (FIE) [Arabidopsis thaliana] 486 19221 CGPG2958 1.00E−159 78 gi|30690446| ref|NP_182182.2|Dof zinc SP finger protein DAG2/Dof affecting germination 2 (DAG2) [Arabidopsis thaliana] 487 70206 CGPG4116 1.00E−139 64 gi|18412918| ref|NP_565249.1|phospholipid/ SP glycerol acyltransferase family protein [Arabidopsis 488 70223 CGPG53 0 93 gi|15240313| ref|NP_198006.1|hexose SP transporter, putative [Arabidopsis thaliana] 489 70347 CGPG3147 1.00E−121 66 gi|18416267| ref|NP_567693.1|Dof-type SP zinc finger domain- containing protein [Arabidopsis thaliana] 490 70406 CGPG1687 0 93 gi|18397470| ref|NP_564354.1|early- SP responsive to dehydration stress protein (ERD4) [Arabidopsis thaliana] 491 70469 CGPG3791 1.00E−171 89 gi|15237581| ref|NP_198936.1|MADS- SP HS box family protein [Arabidopsis thaliana] 492 70564 CGPG1864 0 89 gi|15219067| ref|NP_173589.1|SWIRM SP domain-containing protein/ DNA-binding family protein gb|AAD41423.1| Contains similarity to gb|AF033823 moira protein from Drosophila melanogaster and contains a PF|00249 Myb-like DNA-binding domain. 493 70601 CGPG2917 0 91 gi|15235140| ref|NP_193702.1|zinc finger SP PP (C3HC4-type RING finger) family protein [Arabidopsis thaliana) pir||T04748 hypothetical protein T16H5.30-Arabidopsis thaliana 494 70612 CGPG3721 0 96 gi|18416732| ref|NP_568256.1|conserved SP oligomeric Golgi complex component-related/COG complex component-related [Arabidopsis thaliana] 495 70720 CGPG1358 0 93 gi|15238483| ref|NP_198387.1|lectin SP protein kinase family protein [Arabidopsis thaliana] 496 70735 CGPG2661 1.00E−109 100 gi|15231241| ref|NP_187953.1|transcription SP initiation factor IID-1 (TFIID-1)/TATA-box factor 1/TATA sequence-binding protein 1 (TBP1) [Arabidopsis thaliana] 497 70846 CGPG377 1.00E−151 100 gi|15221223| ref|NP_177577.1|zinc finger SP (C3HC4-type RING finger) family protein [Arabidopsis thaliana] pir||D96772 probable RING zinc finger protein 498 70923 CGPG4020 0 87 gi|8132347| gb|AAF73257.1|MAP SP kinase PsMAPK2 [Pisum sativum] 499 71149 CGPG3457 0 83 gi|20141566| sp|P48001|HKL4_ARATH SP Homeobox protein knotted-1 like 4 (KNAT4) pir||T51795 HOMEOBOX PROTEIN KNOTTED-1 LIKE 4 (KNAT4)- 500 71608 CGPG4687 0 100 gi|15220438| ref|NP_172008.1|ent- SP kaurenoic acid hydroxylase (KAO1)/cytochrome P450 88A3, putative (CYP88A3) [Arabidopsis thaliana] 501 71739 CGPG4345 7.00E−89 80 gi|18406944| ref|NP_566061.1|expressed SP protein [Arabidopsis thaliana] 502 72014 CGPG5230 0 100 gi|25410898| pir||D84423probable WD- SP 40-repeat protein [imported]- Arabidopsis thaliana gb|AAD14533.1| putative stress protein [Arabidopsis thaliana] 503 72051 CGPG5241 0 93 gi|18401606| ref|NP_566585.1|cyclic SP nucleotide-binding transporter 1/CNBT1 (CNGC20) [Arabidopsis thaliana] sp|Q9LD37]CG20_ARATH Probable cyclic nucleotide- gated ion channel 20, chloroplast precursor (Cyclic nucleotide-binding transporter 1) 504 74259 CGPG5343 0 96 gi|15222882| ref|NP_175431.1|branched- CS HS SS chain amino acid aminotransferase 6/ branched-chain amino acid transaminase 6 (BCAT6) [Arabidopsis thaliana] s 505 72463 CGPG4760 8.00E−48 100 gi|15236351| ref|NP_193115.1|auxin- CS SS HS LN PP responsive protein, putative [Arabidopsis thaliana] 506 72902 CGPG5597 0 88 gi|15240576| ref|NP_199800.1|chloride SS CS DS channel protein (CLC-c) [Arabidopsis thaliana] sp|Q96282|CLCC_ARATH Chloride channel protein CLC-c (AtCLC-c) 507 74572 CGPG6640 1.00E−109 93 gi|16331001| ref|NP_441729.1|unknown CS PP SS protein [Synechocystis sp. PCC 6803] 508 73055 CGPG5768 0 97 gi|6321588| ref|NP_011665.1|Hypothetical SS CS HS ORF; Ygr149wp [Saccharomyces cerevisiae] 509 74103 CGPG6558 0 99 gi|15833050| ref|NP_311823.1|fructose- HS PP SS bisphosphate aldolase class II [Escherichia coli O157:H7] r 510 72921 CGPG5781 0 93 gi|6322892| ref|NP_012965.1|general CK PEG SS amino acid permease; Gap1p [Saccharomyces cerevisiae] 511 72968 CGPG5772 0 99 gi|6321546| ref|NP_011623.1|role in PEG LL SS DNA replication during S phase; Clb6p [Saccharomyces cerevisiae] 512 19703 CGPG4172 0 83 gi|7488676| pir||T07150G-box binding HS SS factor 2A-soybean (fragment) gb|AAB00097.1| G-box binding factor 513 19946 CGPG4097 1.00E−46 34 gi|15219099| ref|NP_175691.1|2- SS oxoglutarate-dependent dioxygenase, putative [Arabidopsis thaliana] 514 19980 CGPG3914 2.00E−63 49 gi|28629811| gb|AA045179.1|transcription CS SS factor Myb1 [Malus xiaojinensis] 515 70435 CGPG3701 1.00E−150 91 gi|15236597| ref|NP_193499.1|casein SS PP SP kinase II beta chain, putative [Arabidopsis thaliana] 516 71114 CGPG1657 0 88 gi|30680729| ref|NP_849990.1|K + efflux SS antiporter, putative (KEA4) [Arabidopsis thaliana] 517 72451 CGPG4733 0 94 gi|15239622| ref|NP_197992.1|mitochon- SS drial substrate carrier family protein [Arabidopsis thaliana] 518 72947 CGPG5607 3.00E−62 53 gi|1483230| emb|CAA67968.1|MADS4 SS protein [Betula pendula] 519 73012 CGPG5786 0 97 gi|6324187| ref|NP_014257.1|belongs SS to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NH(4)(+) + NH(3)).; Mep2p [Saccharomyces cerevisiae] 520 73022 CGPG5622 0 86 gi|15225518| ref|NP_182083.1|protein SS kinase family protein [Arabidopsis thaliana] 521 73488 CGPG6394 1.00E−154 94 gi|16080620| ref|NP_391447.1|UTP- SS CS PP glucose-1-phosphate uridylyltransferase [Bacillus subtilis] 522 73901 CGPG5237 0 92 gi|18400284| ref|NP_565553.1|extra- SS large guanine nucleotide binding protein/G-protein (XLG) 523 73964 CGPG5804 0 88 gi|6319773| ref|NP_009855.1|Na+/Pi SS cotransporter, active in early growth phase; similar to phosphate transporters of Neurospora crassa; transcription regulated by inorganic phosphate concentrations and Pho4p; Pho89p [ 524 74019 CGPG5706 2.00E−92 100 gi|16079815| ref|NP_390639.1|adenine SS phosphoribosyltransferase [Bacillus subtilis] 525 74022 CGPG5724 0 97 gi|18378991| ref|NP_563659.1|glycosyl SS SP hydrolase family 3 protein [Arabidopsis thaliana] 526 74114 CGPG6551 0 99 gi|15888903| ref|NP_354584.1|AGR_C_ SS 2921p [Agrobacterium tumefaciens str. C58] pir||H97551 probable aminotransferase aatc 527 74262 CGPG5353 0 100 gi|18416245| ref|NP_568226.1|histidinol- SS PP phosphate aminotransferase, putative [Arabidopsis thaliana] 528 74292 CGPG5367 0 96 gi|15239204| ref|NP_201393.1|U-box SS domain-containing protein [Arabidopsis thaliana] 529 74302 CGPG5384 1.00E−59 82 gi|25313155| pir||A96787protein F10A5.6 PP SS [imported]-Arabidopsis thaliana 530 74325 CGPG5898 1.00E−174 86 gi|15230382| ref|NP_188576.1|cinnamyl- SS alcohol dehydrogenase (CAD) [Arabidopsis thaliana] 531 74429 CGPG6689 0 96 gi|16077873| ref|NP_388687.1|acetoin SS dehydrogenase E1 component (TPP- dependent alpha subunit) [Bacillus subtilis] 532 74440 CGPG6682 6.00E−90 82 gi|15613838| ref|NP_242141.1|uridine SS kinase [Bacillus halodurans C-125] 533 74462 CGPG6668 5.00E−67 99 gi|16332127| ref|NP_442855.1|unknown HS SS protein [Synechocystis sp. PCC 6803] 534 74465 CGPG6692 1.00E−119 99 gi|16078642| ref|NP_389461.1|similar to PP SS ribulose-5-phosphate 3- epimerase [Bacillus subtilis] 535 74474 CGPG6669 2.00E−85 82 gi|16331209| ref|NP_441937.1|unknown LL SS protein [Synechocystis sp. PCC 6803] 536 74505 CGPG6783 0 100 gi|16129426| ref|NP_415984.1|cryptic SS nitrate reductase 2 beta subunit [Escherichia coli K12] 537 74507 CGPG6799 0 82 gi|27479656| gb|AAO17183.1|Orf17 SP SS [Photorhabdus luminescens] 538 74562 CGPG6764 0 95 gi|16077501| ref|NP_388315.1|similar to SS pyruvate oxidase [Bacillus subtilis] Please note that this file doesn't have Table 3.

Screens for Identifying Trait Improving Genes

DS—Improvement of Drought Tolerance Identified by a Soil Drought Stress Tolerance Screen:

Drought is a water deficit condition that imposes osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The drought tolerance-imparting DNA defined for this invention are used in recombinant DNA constructs that improve plant survival rate under drought conditions. Exemplary recombinant DNA which has been identified for conferring such drought tolerance is identified as such in Table 2. Such identified recombinant DNA is useful in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.

PEG—Improvement of Drought Tolerance Identified by PEG Induced Osmotic Stress Tolerance Screen:

Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits et al., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. Certain embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen survive drought conditions providing a higher yield potential as compared to control plants.

SS—Improvement of Drought Tolerance Identified by High Salinity Stress Tolerance Screen:

Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. Exemplary recombinant DNA which has been identified to help plants maintain biomass, root growth and/or plant development in high salinity conditions are identified as such in Table 2. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, embodiments of trait-improving recombinant DNA identified in a high salinity stress tolerance screen also provide transgenic crops with improved drought tolerance. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen survive drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.

HS—Improvement of Drought Tolerance Identified by Heat Stress Tolerance Screen:

Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.

CK and CS—Improvement of Tolerance to Cold Stress:

Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. Two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8 degrees C. from planting until day 28 post planting. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22 degrees C. until day 8 post planting, and subsequently were placed under 8 degrees C. until day 28 post planting. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen survive cold conditions providing a higher yield potential as compared to control plants.

Improvement of Tolerance to Multiple Stresses:

Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, plants are enabled to react to different kinds of stresses. For examples, DNA for expressing proteins of SEQ ID NO:352 and SEQ ID NO:353 is useful to improve both heat stress tolerance and cold stress tolerance in plants. Plants transformed with DNa for expressing protein of SEQ ID NO:508 resist heat stress, salt stress and cold stress. Thus, the disclosed stress tolerance conferring genes are useful in combinations to generate transgenic plants that resist multiple stress conditions.

PP—Improvement of Early Plant Growth and Development:

It is known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis; a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems, that continuously generate new organs throughout post-embryonic development. “Early growth and development” encompasses the stages of seed imbibition through the early vegetative phase. Certain DNA is identified as useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, genes conferring the growth advantage in early stages to plants are used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. Exemplary recombinant DNA that confers both stress tolerance and growth advantages to plants, is identified as such in Table 2, e.g., DNA encoding a protein of SEQ ID NO:444 can improve the plant early growth and development, and impart heat and cold tolerance to plants. Embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.

SP—Improvement of Late Plant Growth and Development:

“Late growth and development” encompasses the stages of leaf development, flower production, and seed maturity. Transgenic plants with late growth and development advantages express DNA that is identified as such in Table 2. Such plants exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. For example, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. Seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. Embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.

LL—Improvement of Tolerance to Shade Stress Identified in a Low Light Screen:

The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. Recombinant DNA that enables plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently provides embodiments of those plants with higher yield as compared to the wild type plants. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by this invention are suitable for a higher density planting, thereby resulting increased yield per unit area.

LN—Improvement of Tolerance to Low Nitrogen Availability Stress

Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.

Recombinant DNA that imparts enhanced nitrogen use efficiency in transgenic plants is identified in Table 2. Such plants exhibit one or more desirable traits including, but not limited to, increased seedling weight, increased number of green leaves, increased number of rosette leaves, altered root length and advanced flower bud formation. Such plants can also have altered amino acid or protein compositions, increased yield and/or better seed quality. Embodiments of such transgenic plants are productively cultivated under nitrogen nutrient deficient conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs that cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless under such conditions. The transgenic plants also are advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.

Stacked Traits:

This invention also provides transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a glyphosate resistance trait, or insect resistance, such as using a gene from Bacillus thuringiensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. 2003-0106096 A1 and 2002-0112260 A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.

Once one recombinant DNA has been identified as conferring an improved trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant trait-imparting DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant and different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, this invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO:1 through SEQ ID NO:269, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO:270 through SEQ ID NO:538. In another aspect, this invention provides a consensus amino acid sequence for respective homologs for each of SEQ ID NO:270 through SEQ ID NO:538. In yet another aspect, this invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence disclosed herein.

The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by this invention are not limited to any particular plant species. Indeed, the plants of this invention encompass many species of monocots and dicots and include agriculturally useful plants which are cultivated for purposes of food production or industrial applications, e.g., corn and soybean plants and cotton plants. Recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are disclosed in the following examples. Other plants of this invention include canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

Thus, embodiments of this invention include the use of both DNA identified in Table 3 and homologs in recombinant DNA for transgenic crop plants with improved traits. Transgenic crop plants with improved traits are identified from populations of plants grown from transgenic events by screening to segregate the plants of this invention from plants without the improved traits. Preferred screens for transgenic crop plants identify plants with improved responses to stress conditions, e.g., assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures are made on greenhouse or field grown plants and include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.

Trait data on morphological changes is collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits are identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barrenness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain are confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

To confirm hybrid yield in transgenic corn plants expressing trait-imparting DNA of this invention, it is useful to test hybrid plants over multiple years at multiple locations in a geographical location where corn is conventionally grown, e.g., in Iowa, Illinois and Kansas, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.

Transgenic crop plants are used to provide other aspects of this invention such as transgenic seeds of crop plants. Seeds of transgenic plants are used to propagate more progeny plants which contain the trait-improving recombinant DNA constructs of this invention. These progeny plants are within the scope of this invention when they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.

Screening Methods for Crop Transgenic Plants with Enhanced Agronomic Trait

Due to variability in transformation many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. Thus, screening is necessary to identify the transgenic events that produce the transgenic plants and seeds of this invention. Transgenic crop plants having enhanced traits are identified from populations of plants transformed as described herein by evaluating the trait in a variety of assays to detect an enhanced agronomic trait. Useful assays include analyses to detect changes in the chemical composition, biomass, physiological properties and morphology of the plant. Changes in chemical compositions such as nutritional composition of grain are detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics are detected in greenhouse or field grown plants and include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties are identified by evaluating responses to stress conditions, e.g., assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology are measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other screening properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barrenness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain are evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.

Seeds for transgenic crop plants with enhanced agronomic traits of this invention are corn, soybean and cotton seeds, as well as seeds for canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

A. Screening for Nitrogen Use Efficiency

Many transgenic crop plants of this invention exhibit enhanced nitrogen use efficiency as compared to control plants. Higher nitrogen soil applications increase seed protein and starch accumulation, and lead to larger seed weight and larger kernel number per ear. Recent improvements in elite high yielding corn hybrid genotypes include the ability to utilize nitrogen efficiently. DNA causing the enhanced nitrogen use efficiency in crop plants are especially useful, e.g., for improving yield. Enhanced nitrogen use efficiency is assessed by measuring changes in plant growth such as leaf area production, shoot biomass, chlorophyll content in plants grown in nitrogen limiting conditions and/or nitrogen sufficient conditions. It is useful to conduct a first screen in nitrogen limiting conditions and confirm replicate transgenic events in both nitrogen limiting and nitrogen sufficient conditions. Table 4 shows an amount of nutrients in the nutrient solution for nitrogen limiting conditions (low N) and nitrogen sufficient conditions (high N) which are useful for nitrogen use efficiency screening. For example in a greenhouse screen pots of transgenic plants and control plants are treated with 100 ml of nutrient solution three times a week on alternate days starting at 8 and 10 days after planting for high N and low N screening, respectively.

TABLE 4 2 mM NH₄NO₃ 20 mM NH₄NO₃ Nutrient stock Low nitrogen High nitrogen 1M NH₄NO₃ 2 mL/L 20 mL/L 1M KH₂PO₄ 0.5 0.5 1M MgSO₄•7H₂O 2 2 1M CaCl₂ 2.5 2.5 1M K₂SO₄ 1 1 Note: Adjust pH to 5.6 with HCl or KOH After 28 days of plant growth for low N screening and 23 days for high N screening, measurements are taken for total shoot fresh mass, leaf chlorophyll, leaf area, leaf fresh mass and leaf dry mass.

B. Screening for Increased Yield

Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from a variety or other traits such as enhanced seed sink potential, e.g., the number and size of endosperm cells or kernels, and/or enhanced sink strength, e.g., the rate of starch biosynthesis. Sink potential is established very early during kernel development, as endosperm cell number and cell size are determined within the first few days after pollination.

Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.

Effective yield screening of transgenic corn uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Useful screening in multiple and diverse geographic locations, e.g., up to 16 or more locations, over one or more planting seasons, e.g., at least two planting seasons, is useful to statistically distinguish yield improvement from natural environmental effects. Useful hybrid screening includes planting multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, e.g., 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Plants from separate transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre.

Surrogate indicators for screening for yield improvement include source capacity (biomass), source output (sucrose and photosynthesis), sink components (kernel size, ear size, starch in the seed), development (light response, height, density tolerance), maturity, early flowering trait and physiological responses to high density planting, e.g., at 45,000 plants per acre.

When screening for yield improvement a useful statistical measurement approach comprises three components, i.e., modeling spatial autocorrelation of the test field separately for each location, adjusting traits of recombinant DNA events for spatial dependence for each location, and conducting an across location analysis.

A first step in modeling spatial autocorrelation is estimating the covariance parameters of the semivariogram. A spherical covariance model is assumed to model the spatial autocorrelation. Because of the size and nature of the trial, it is likely that the spatial autocorrelation may change. Therefore, anisotropy is also assumed along with spherical covariance structure. The following set of equations describes the statistical form of the anisotropic spherical covariance model.

${{C\left( {h;\theta} \right)} = {{{vI}\left( {h = 0} \right)} + {{\sigma^{2}\left( {1 - {\frac{3}{2}h} + {\frac{1}{2}h^{3}}} \right)}{I\left( {h < 1} \right)}}}},$

where I(•) is the indicator function, h=√{square root over ({dot over (x)}²+{dot over (y)}²)}, and

{dot over (x)}=[cos(ρπ/180)(x ₁ −x ₂)−sin(ρπ/180)(y ₁ −y ₂)]/ω_(x)

{dot over (y)}=[sin(ρπ/180)(x ₁ −x ₂)+cos(ρπ/180)(y ₁ −y ₂)]/ω_(y)

where s₁=(x₁, y₁) are the spatial coordinates of one location and s₂=(x₂, y₂) are the spatial coordinates of the second location. There are 5 covariance parameters, θ=(ν,σ⁻²,ρ,ω_(n),ω_(j)), where ν is the nugget effect, σ² is the partial sill, ρ is a rotation in degrees clockwise from north, ω_(n) is a scaling parameter for the minor axis and ω_(j) is a scaling parameter for the major axis of an anisotropical ellipse of equal covariance. The five covariance parameters that define the spatial trend will then be estimated by using data from heavily replicated pollinator plots via restricted maximum likelihood approach. In a multi-location field trial, spatial trend are modeled separately for each location.

After obtaining the variance parameters of the model, a variance-covariance structure is generated for the data set to be analyzed. This variance-covariance structure contains spatial information required to adjust yield data for spatial dependence. In this case, a nested model that best represents the treatment and experimental design of the study is used along with the variance-covariance structure to adjust the yield data. During this process the nursery or the seed batch effects can also be modeled and estimated to adjust the yields for any yield parity caused by seed batch differences.

After spatially adjusted data from different locations are generated, all adjusted data is combined and analyzed assuming locations as replications. In this analysis, intra and inter-location variances are combined to estimate the standard error of yield from transgenic plants and control plants. Relative mean comparisons are used to indicate statistically significant yield improvements.

C. Screening for Water Use Efficiency

Many transgenic crop plants of this invention exhibit improved yield resulting from improved water use efficiency and/or drought tolerance.

A greenhouse screen for transgenic corn plants for water use efficiency measures changes in plant growth rate, e.g., at least a 10% improvement, in height and biomass during a vegetative drought treatment, as compared to control plants. The hydration status of the shoot tissues following the drought is also measured. Shoot Initial Height (SIH) is plant height after 3 weeks of growth under optimum conditions. Shoot Wilt Height (SWH) is plant height at the end of a 6 day drought. Time course experiments have shown that at about 3 days of drought, wild type plants basically stop growing and begin to wilt. Thus a transgenic plant with improved water use efficiency will continue to grow (probably to a lesser extent than with water) and thereby end up as a significantly taller plant at the end of a drought experiment. Shoot Wilt Mass (SWM) is the amount of wet and dry matter in the shoot (plant separated from root ball at the soil line) at the end of the drought; SDM is measure after 2 to 3 weeks in a drying chamber. Shoot Turgid mass (STM) is the SWM plus the mass of the water that is transported into plant tissues in 3 days of soaking in 40 degree C. water in the dark. Experiments show that most of the water is pulled up in 24 hours but it takes 2 more days before additional increase becomes insignificant. STM-SWM is indicative of water use efficiency in plants where recovery from stress is more important than stress tolerance per se. Relative water content (RWC) is a measurement of how much (%) of the plant is water at harvest. RWC=(SWM−SDM)/(STM−SDM)*100. Fully watered corn plants are about 98% RWC. Typically, in a wilt screen the plants are about 60% RWC. Plants with higher RWC at the end of a drought are considered to be healthier plants and more fit for post-drought recovery and growth.

Relative Growth Rate (RGR) is calculated for each shoot using the formula RGR=(SWH−SIH)/((SWH+SIH)/2)*100

D. Screening for Growth Under Cold Stress

Many transgenic crop plants of this invention exhibit improved growth under cold stress, e.g., in a cold germination assay, in a cold shock assay, in an early seedling growth assay and in root-shoot biomass assay.

In a cold germination assay transgenic seeds from transgenic plants, e.g., R2 inbred seeds or F1 hybrid seeds, seeds of two types of control plants, e.g., negative segregants from the transgenic event or wild type, non-transgenic seeds of the transformed genotype, are treated with fungicide. A useful fungicide such as Captan fungicide (available from Arvesta Corp as MAESTRO® 80DF Fungicide) is applied at the rate of 0.43 mL Captan per 45 g of corn seeds which are dried to provide fungicide-coated seeds.

In a useful cold screen for transgenic corn seeds ten seeds per transgenic event are placed on filter paper (e.g., Whatman No. 1) in the lid of a Petri dish with 5 ml of water. A closed Petri dish is placed in a growth chamber set at 11 degrees C. for inbred corn seed or 9.5 degrees C. for hybrid corn seed. 2 ml of water is added on day 3 and day 10. Seeds are considered germinated if the emerged radicle size is 1 cm. Cold seeds are scored every 2 days from day 10 up to day 30. Tissue samples are collected at random on the last day of the experiment for confirmation of RNA expression. A germination index (GI) is calculated as

GI=(Σ([T+1−n _(i) ]*[P _(i) −P _(i-1)]))/T

where “T” is the number of days for the experiment, “n” is the number of days after start, “i” is number of times germination is counted including the current day, “P” is the percentage of seed germinated during any given rating. Statistical differences are calculated between positive and wild type control.

In a cold shock assay, seeds are planted in potting media and placed in a growth chamber set at 23 degrees C., relative humidity of 65% with 12 hour day and night photoperiod (300 uE/m2-min). Planted seeds are watered for 20 minute every other day by sub-irrigation and flats are rotated every third day. On day 10 after planting the transgenic positive and wild type control plants are positioned in flats in an alternating pattern. Chlorophyll fluorescence of plants is measured on the tenth day during the dark period of growth by using a Walz PAM-2000 portable fluorometer following manufacturer's instructions. After chlorophyll measurements, leaf samples from each event are collected for confirming the expression of recombinant DNA. The plants are then exposed to temperatures of 5 degrees C. for 4 days. On the fourth day chlorophyll fluorescence is measured and plants are restored to a 23 degrees C. environment for recovery over 3 days. During the recovery period the length of the V3 leaf is measured on the first and third days. After two days of recovery V2 leaf damage is determined visually by estimating percent of green V2 leaf. Statistical differences in V3 leaf growth, V2 leaf necrosis and fluorescence during pre-shock and cold shock can be used for estimation of cold shock damage on corn plants.

In an early seedling growth assay three sets of seeds are assayed. The first set is a group of transgenic seeds from transgenic plants; the second set is negative segregants of the transgenic seed; and the third seed set is seed from two cold tolerant and two cold sensitive wild-type controls. All seeds are treated with a fungicide as indicated above. Seeds are grown in germination paper (12 inch×18 inch pieces of Anchor Paper #SD7606), wetted in a solution of 0.5% KNO₃ and 0.1% Thyram. For each paper fifteen seeds are placed on the line evenly spaced such that the radicles will grow toward the same edge. The wet paper is rolled up evenly and tight enough to hold the seeds in place. The roll is secured into place with two large paper clips, one at the top and one at the bottom. The rolls are incubated in a growth chamber at 23 degrees C. for three days in a randomized complete block design within an appropriate container. The chamber is set for 65% humidity with no light cycle. For the cold stress treatment the rolls are then incubated in a growth chamber at 12 degrees C. for fourteen days. The chamber is set for 65% humidity with no light cycle. For the warm treatment the rolls are incubated at 23 degrees C. for an additional two days. After the treatment the germination papers are unrolled and the seeds that did not germinate are discarded. The lengths of the radicle and coleoptile for each seed are measured. A coleoptile sample is collected from six individual kernels of each entry for confirming the expression of recombinant DNA. Statistical differences in the length of radicle and shoot during pre-shock and cold shock are used for an estimation of the effect of the cold treatment on corn plants. The analysis is conducted independently for the warm and cold treatments.

In a root-shoot biomass assay two sets of seeds are used. The first set is transgenic seeds with recombinant DNA, e.g., R2 inbred seeds or F1 hybrid seeds; the second seed set is non-transgenic, wild type negative control made from the same genotypes as the transgenic seeds. All seeds are treated with a fungicide as indicated above. The seeds are planted in potting media in pots arranged in a randomized complete block design with 6 replications. Pots are watered as and when needed by filling water up to the brim of the pot. Plants are grown in a greenhouse to a V6 stage or approximately for 28 days. Greenhouse lights are turned on after emergence of seedlings with 14 hours of light 10 hours of dark. Plants are fertilized twice each week with water-soluble fertilizer containing 200-ppm nitrogen. For measurement of root and shoot dry weight, two pots are separated carefully to remove adhering sand by washing with water. Washed roots are cut at the first node. The roots are placed in a paper bag after squeezing excess water, folded once and stapled. The shoots are then folded up to a convenient size (approximately 15 cm), placed in a paper bag. Bags are placed over a wire shelve to facilitate drying in a ventilated room maintained at 120 degrees F. to a moisture content of about 13% then weighed to determine dried root and shoot biomass.

E. Screen for Enhanced Oil, Starch, or Protein Levels in Plant Seeds

Oil concentrations are determined in kernels by Near Infrared Transmittance (NIT) from inbred and from hybrid lines. Data are also obtained for protein and starch content from this measurement.

Inbred Kernel Oil Screen

The primary transformants are selfed to produce RI seed which is planted to segregating seed. An untransformed control line is planted every sixth row. All plants are self-pollinated. A molecular assay is conducted to determine zygosity of the transgene in each plant. Ears are harvested at maturity, and well-filled ears are chosen for proximate analysis. Proximate analysis is conducted on up to 5 homozygous ears. If 5 good homozygous ears are not available, then hemizygous ears will be used to obtain 5 good transgene-positive ears. Statistical analysis is conducted to determine whether proximate values for transgenic events are different from controls. Events with an increase in oil with a p-value of less than or equal to 0.1 are termed “putative leads.” Kernel composition is confirmed in an inbred confirmation nursery which is conducted with selected events, and is run with a design similar to that of the Gen2 nursery. A “confirmed lead event” demonstrates an increase in oil with a p-value of less than or equal to 0.1 in two nurseries.

Hybrid Kernel Oil Screen

Grain samples from the multilocation hybrid yield trials are collected at the time of harvest and are analyzed by NIT. Controls are negative segregants, untransformed controls, or pollinators. Data from 3 to 12 locations are pooled for the statistical analysis. Putative leads have increased oil with a p-value of less than or equal to 0.1.

The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.

Example 1 Identification of Recombinant DNA that Confers Improved Trait(s) to Plants A. Expression Constructs for Arabidopsis Plant Transformation

Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, et al., PNAS 95:376-381, 1998; Guyer, et al., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, et al., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.

B. Soil Drought Tolerance Screen

This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions

T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m²/s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.

To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.

At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.

Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). Table 5 provides a list of recombinant DNA constructs that improve drought tolerance in transgenic plants.

TABLE 5 Survival Anaysis of Wilt Response Seed Weight/ wilt response Pep Risk score plant diff SEQ Con- Orienta- RS p- p- time to p- ID struct_id Gene tion mean value c delta value c wilting value c 319 10139 CGPG101 ANTI- 0.115 0.024 S −0.123 0.804 / −0.01 0.469 / SENSE 320 11410 CGPG103 SENSE 0.226 0.003 S −0.366 0.926 / 0 1 / 321 11604 CGPG48 ANTI- 0.25 0.034 S 0.257 0.01 S −0.24 0.38 / SENSE 322 12368 CGPG1006 SENSE 0.148 0.044 S 0.359 0.02 S −0.06 0.764 / 323 13502 CGPG1354 SENSE 0.431 0 S 0.624 0 S 1.34 0.366 / 324 13745 CGPG1576 ANTI- −0.021 0.711 / 0.664 0 S −0.29 0.453 / SENSE 325 13821 CGPG1569 SENSE 0.135 0.025 S 0.128 0.402 / 0.24 0.972 / 326 14240 CGPG1697 SENSE 0.377 0.002 S −1.305 0.991 / 0 1 / 327 14718 CGPG1082 SENSE 0.168 0.001 S 0.135 0.351 / 0.25 0.208 / 328 17022 CGPG1774 SENSE 0.06 0.124 T 0.563 0.043 S 0 0.961 / 329 17924 CGPG2882 SENSE −0.093 0.914 / 0.288 0.021 S 0.09 0.935 / 330 18259 CGPG3368 SENSE 0.07 0.28 / 0.391 0.058 T −0.27 0.591 / 331 19171 CGPG2952 SENSE 0.227 0.005 S 0.846 0.001 S 0.12 0.543 / 332 19201 CGPG2332 SENSE 0.124 0.027 S −0.435 0.785 / 0.35 0.256 / 333 19317 CGPG3662 SENSE 0.338 0 S −0.071 0.61 / 0.63 0.106 T 334 70417 CGPG3427 SENSE 0.253 0.016 S −1.424 0.984 / 0 1 / 315 70427 CGPG3067 SENSE −0.033 0.818 / 1.004 0.002 S 0.01 0.977 / 335 70467 CGPG3785 SENSE 0.127 0.023 S −0.448 0.946 / 0.71 0.046 S 336 70806 CGPG712 SENSE 0.246 0.046 S 0.174 0.276 / 0.14 0.07 T 337 70818 CGPG479 SENSE 0.07 0.115 T 0.558 0.009 S 0.61 0.283 / 338 70820 CGPG655 SENSE 0.172 0.048 S 0.036 0.441 / 0.26 0.554 / 339 70919 CGPG4029 SENSE 0.167 0.009 S −0.565 0.904 / 0.31 0.508 / 340 71623 CGPG4696 SENSE 0.158 0.047 S 0.421 0.04 S 0 1 / 390 71633 CGPG857 SENSE 0.121 0.017 S −0.823 0.967 / 0.45 0.139 T 341 71662 CGPG4679 SENSE 0.063 0.013 S −0.037 0.631 / 0.16 0.957 / 342 71693 CGPG4652 SENSE 0.074 0.042 S 0.246 0.06 T 0.34 0.616 / 316 71811 CGPG4426 SENSE 0.359 0.015 S −0.729 0.903 / 0.15 0.822 / 318 72081 CGPG5279 SENSE 0.269 0.005 S −0.372 0.987 / 0.17 0.404 / 343 72384 CGPG4639 SENSE 0.133 0.018 S 0.62 0.002 S 0.17 0.359 / 344 72439 CGPG5075 SENSE 0.175 0.013 S −0.035 0.604 / 0.23 0.244 / 374 72456 CGPG4745 SENSE 0.53 0.01 S −0.737 0.979 / −0.08 0.823 / 345 72619 CGPG4835 SENSE 0.178 0.039 S 0.219 0.072 T 0.96 0.691 / 346 72624 CGPG4842 SENSE 0.163 0.021 S 0.356 0.051 T 0.3 0.375 / 347 72715 CGPG5521 SENSE 0.082 0.1 T 0.767 0.002 S 0.12 0.628 / 348 72754 CGPG5548 SENSE 0.131 0.031 S −0.365 0.974 / 0 0.923 / 349 72819 CGPG4989 SENSE 0.094 0.026 S 0.362 0.069 T 0.03 0.83 / 350 75516 CGPG7689 SENSE 0.067 0.066 T 0.965 0.001 S 0.24 0.464 / 351 75701 CGPG7856 SENSE 0.147 0.006 S 0.986 0.015 S 0.15 0.448 / 317 73463 CGPG6384 SENSE 0.174 0.048 S −1.359 0.959 / 0.09 0.984 / 454 70354 CGPG3995 SENSE −0.005 0.563 / 0.444 0.002 S 10.29 0.9 / 397 71840 CGPG4353 SENSE 0.142 0.007 S −0.212 0.859 / 9.29 0.99 / 506 72902 CGPG5597 SENSE 0.009 0.162 T 0.034 0.2 T 5 1 / 437 73549 CGPG6460 SENSE 0.119 0.037 S −0.774 0.949 / 6.26 0.25 / 302 73586 CGPG6471 SENSE 0.003 0.451 / 0.588 0.002 S 6.34 0.723 / 442 74587 CGPG6774 SENSE 0.262 0.001 S −0.117 0.574 / 7.49 0.041 S 388 74652 CGPG6168 SENSE 0.475 0 S −0.766 0.92 / 7.48 0 S S: represents that the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05, p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance) T: represents that the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2 /: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset.

C. Heat Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.

T2 seeds were plated on ½×MS salts, V % phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was ˜140 μmol/m²/s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.

The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, et al., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. Table 6 provides a list of recombinant DNA constructs that improve heat tolerance in transgenic plants.

TABLE 6 Seedling Pep Growth stage Weight SEQ Con- Orienta- RS p- Root Length p- ID struct_id Gene tion mean value c delta p-value c delta value c 354 19542 CGPG3069 SENSE 0.842 0.021 S 0.296 0.023 S 1.616 0 S 355 19618 CGPG3574 SENSE 1.072 0.005 S 0.328 0.006 S 1.569 0 S 356 19649 CGPG3140 SENSE 0.72 0.042 S 0.243 0.054 T 1.665 0 S 357 19745 CGPG3973 SENSE 0.65 0.016 S 0.19 0.021 S 1.505 0 S 358 19768 CGPG4096 SENSE 1.06 0.035 S 0.17 0.034 S 1.171 0 S 436 19771 CGPG4011 SENSE 0.822 0.023 S 0.201 0.001 S 1.43 0 S 359 19772 CGPG3939 SENSE 0.247 0.014 S 0.151 0.014 S 1.344 0 S 360 19779 CGPG4113 SENSE 0.605 0.051 T 0.181 0.003 S 1.436 0 S 361 19833 CGPG4074 SENSE 0.965 0.026 S 0.266 0.007 S 1.01 0 S 362 19862 CGPG3961 SENSE 0.341 0.119 T 0.132 0.007 S 1.22 0 S 363 19879 CGPG4009 SENSE 0.734 0.002 S 0.22 0.001 S 1.499 0 S 364 70445 CGPG3728 SENSE 0.413 0.062 T 0.148 0.114 T 1.249 0 S 365 70738 CGPG3195 SENSE 0.687 0.055 T 0.205 0.041 S 1.261 0 S 366 71437 CGPG4043 SENSE 0.094 0.198 T 0.092 0.064 T 1.301 0 S 367 71572 CGPG4520 SENSE 0.938 0.052 T 0.441 0 S 1.633 0 S 368 71617 CGPG1227 SENSE 0.809 0.012 S 0.143 0.029 S 1.05 0.003 S 408 72085 CGPG5228 SENSE 1.234 0.02 S 0.192 0.043 S 1.162 0 S 369 72532 CGPG4780 SENSE 1.028 0.022 S 0.198 0.052 T 1.043 0.001 S 409 72744 CGPG5563 SENSE 0.17 0.146 T 0.04 0.359 / 0.827 0.004 S 370 72757 CGPG5572 SENSE 1.82 0.004 S 0.14 0.091 T 1.121 0 S 407 72771 CGPG2166 SENSE 1.776 0.001 S 0.36 0 S 1.428 0 S 444 72967 CGPG5742 SENSE 0.273 0.063 T 0.147 0.102 T 1.03 0 S 410 73039 CGPG810 SENSE −0.048 0.774 / −0.135 0.957 / 0.59 0.022 S 411 73054 CGPG5754 SENSE 0.055 0.312 / 0.236 0.001 S 1.434 0 S 508 73055 CGPG5768 SENSE 0.154 0.123 T 0.269 0 S 1.524 0 S 371 73412 CGPG6448 SENSE 0.187 0.118 T 0.134 0.06 T 1.181 0 S 412 73501 CGPG6456 SENSE 1.6 0.003 S 0.081 0.136 T 1.119 0 S 352 73515 CGPG6473 SENSE −0.037 0.758 / −0.024 0.604 / 0.694 0.008 S 437 73549 CGPG6460 SENSE 2.612 0 S 0.199 0.017 S 1.432 0 S 372 74102 CGPG6550 SENSE 0.34 0.035 S 0.268 0.002 S 1.355 0 S 509 74103 CGPG6558 SENSE −0.013 1 / −0.021 0.608 / 0.86 0 S 353 74684 CGPG6360 SENSE 0.44 0.018 S 0.254 0.002 S 1.383 0 S 512 19703 CGPG4172 SENSE 0.211 0.079 T 0.059 0.301 / 1.262 0 S 273 70423 CGPG3165 SENSE 1.456 0 S 0.418 0 S 1.912 0 S 491 70469 CGPG3791 SENSE 0.143 0.28 / 0.028 0.349 / 1.001 0.001 S 434 70932 CGPG4089 SENSE 0.354 0.1 T 0.03 0.419 / 1.254 0 S 419 71134 CGPG817 SENSE 0.522 0.106 T 0.077 0.248 / 1.225 0 S 466 71726 CGPG3894 SENSE 0.196 0.225 / 0.082 0.2 T 1.243 0 S 505 72463 CGPG4760 SENSE 1.182 0.003 S 0.202 0.026 / 1.702 0 S 445 72961 CGPG5591 SENSE 1.195 0.013 S 0.106 0.105 T 1.084 0 S 306 74136 CGPG6632 SENSE 0.886 0.012 S 0.306 0.009 S 1.42 0 S 504 74259 CGPG5343 SENSE 1.044 0.023 S 0.081 0.187 T 1.274 0 S 310 74318 CGPG5826 SENSE 0.407 0.075 T 0.116 0.052 T 1.118 0 S 479 74344 CGPG5929 SENSE 1.256 0.018 S 0.118 0.125 T 1.275 0 S 533 74462 CGPG6668 SENSE 0.846 0.022 S 0.26 0.011 S 1.425 0 S 313 74512 CGPG32 SENSE 0.241 0.118 T 0.146 0.044 S 1.062 0 S 442 74587 CGPG6774 SENSE 0 / / 0.084 0.054 T 1.046 0 S S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2 /: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset

D. Salt Stress Tolerance Screen

This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.

T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m². On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.

The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1L. Table 7 provides a list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants

TABLE 7 Seedling Root Length Root Length Weight Pep Growth Stage at day 11 at day 14 at day 14 SEQ Con- Orienta- RS p- p- p- p- ID struct id Gene tion mean value c delta value c delta value c delta vallue c 512 19703 CGPG4172 SENSE 1.124 0.139 T 0.198 0.021 S 0.072 0.116 T 0.582 0.023 S 513 19946 CGPG4097 SENSE 1.201 0.072 T 0.02 0.89 / 0.069 0.573 / 0.443 0.266 / 514 19980 CGPG3914 SENSE 0.904 0.146 T 0.101 0.259 / 0.144 0.058 T 0.706 0.016 S 515 70435 CGPG3701 SENSE 1.363 0.031 S −0.118 0.228 / 0.161 0.038 S 0.053 0.697 / 516 71114 CGPG1657 SENSE 0.138 0.399 / 0.245 0.009 S 0.187 0.025 S 0.472 0.069 T 517 72451 CGPG4733 SENSE 2.226 0.02 S 0.186 0.006 S 0.069 0.23 / 0.466 0.011 S 443 72453 CGPG4735 SENSE 1.539 0.031 S 0.232 0.002 S 0.216 0 S 0.737 0.001 S 505 72463 CGPG4760 SENSE 3.026 0.002 S 0.119 0.269 / 0.202 0.073 T 1.26 0 S 392 72519 CGPG4749 SENSE 0.598 0.041 S 0.126 0.055 T 0.172 0.003 S 0.635 0.008 S 506 72902 CGPG5597 SENSE 1.418 0.039 S 0.114 0.286 / 0.226 0.078 T 0.426 0.091 T 448 72916 CGPG1814 SENSE 0.682 0.181 T 0.07 0.532 / 0.201 0.046 S 0.387 0.079 T 510 72921 CGPG5781 SENSE 1.977 0.029 S 0.163 0.176 T 0.211 0.073 T 0.663 0.006 S 518 72947 CGPG5607 SENSE 1.505 0.028 S 0.024 0.899 / 0.204 0.022 S 0.466 0.216 / 445 72961 CGPG5591 SENSE 1.879 0.007 S 0.228 0.122 T 0.229 0.003 S 0.817 0.04 S 444 72967 CGPG5742 SENSE 2.427 0.004 S 0.386 0.009 S 0.369 0.001 S 1.254 0 S 511 72968 CGPG5772 SENSE 1.531 0.055 T 0.302 0.06 T 0.209 0.073 T 0.761 0.003 S 449 72969 CGPG5789 SENSE 0.67 0.078 T 0.029 0.789 / 0.239 0.008 S 0.603 0.013 S 519 73012 CGPG5786 SENSE 2.371 0.001 S 0.366 0 S 0.342 0 S 1.08 0 S 520 73022 CGPG5622 SENSE 1.408 0.036 S 0.22 0.056 T 0.347 0 S 0.492 0.03 S 508 73055 CGPG5768 SENSE 3.291 0.001 S 0.369 0.087 T 0.417 0.005 S 1.096 0.005 S 446 73070 CGPG5627 SENSE 2.755 0.005 S 0.188 0.392 / 0.275 0.011 S 0.685 0.025 S 447 73475 CGPG6385 SENSE 1.11 0.059 T 0.127 0.361 / 0.216 0.001 S 0.474 0.06 T 521 73488 CGPG6394 SENSE 2.373 0.013 S 0.314 0.006 S 0.262 0.002 S 1.126 0.005 S 522 73901 CGPG5237 SENSE 1.141 0.078 T 0.207 0.197 T 0.202 0.097 T 0.639 0.03 S 523 73964 CGPG5804 SENSE 1.235 0.043 S 0.428 0.008 S 0.317 0.022 S 0.955 0.002 S 524 74019 CGPG5706 SENSE 0.105 0.168 T 0.074 0.649 / 0.171 0.106 T 0.773 0.011 S 525 74022 CGPG5724 SENSE 0.033 0.327 / −0.065 0.616 / 0.172 0.032 S 0.484 0.068 T 509 74103 CGPG6558 SENSE 1.225 0.074 T 0.26 0.042 S 0.267 0.004 S 0.543 0.042 S 526 74114 CGPG6551 SENSE 3.627 0 S 0.265 0.119 T 0.26 0 S 0.561 0.063 T 504 74259 CGPG5343 SENSE 2.802 0.003 S 0.249 0.098 T 0.256 0.037 S 0.995 0 S 527 74262 CGPG5353 SENSE 0.225 0.319 / 0.238 0.062 T 0.247 0 S 0.629 0.006 S 528 74292 CGPG5367 SENSE 0.327 0.199 T 0.16 0.067 T 0.105 0.166 T 0.565 0.013 S 529 74302 CGPG5384 SENSE 1.246 0.016 S 0.296 0.004 S 0.25 0 S 0.705 0.004 S 530 74325 CGPG5898 SENSE 1.596 0.021 S −0.035 0.76 / 0.094 0.106 T 0.685 0.004 S 531 74429 CGPG6689 SENSE 1.796 0.008 S 0.298 0.037 S 0.207 0.006 S 0.496 0.029 S 532 74440 CGPG6682 SENSE 0.223 0.334 / 0.43 0.007 S 0.272 0.01 S 0.744 0.017 S 450 74449 CGPG6659 SENSE 0.693 0.19 T 0.204 0.104 T 0.205 0.022 S 0.451 0.095 T 533 74462 CGPG6668 SENSE 2.14 0.028 S 0.244 0.038 S 0.239 0.001 S 0.64 0.013 S 534 74465 CGPG6692 SENSE 1.245 0.016 S 0.35 0.01 S 0.215 0.001 S 0.575 0.043 S 535 74474 CGPG6669 SENSE 3.312 0.002 S 0.233 0.083 T 0.338 0.003 S 0.589 0.044 S 536 74505 CGPG6783 SENSE 1.731 0.043 S 0.272 0.007 S 0.208 0.01 S 0.493 0.009 S 537 74507 CGPG6799 SENSE 2.32 0.009 S 0.056 0.567 / 0.227 0.035 S 0.476 0.126 T 538 74562 CGPG6764 SENSE 1.405 0.038 S −0.052 0.776 / 0.215 0.014 S 0.104 0.766 / 507 74572 CGPG6640 SENSE 1.425 0.025 S 0.201 0.009 S 0.267 0.001 S 1.184 0 S 270 14324 CGPG1560 SENSE 1.708 0.017 S 0.307 0.01 S 0.408 0 S 0.995 0.002 S 358 19768 CGPG4096 SENSE 1.496 0.061 T 0.163 0.187 T 0.129 0.013 S 0.66 0.002 S 436 19771 CGPG4011 SENSE 1.666 0.051 T 0.319 0.005 S 0.201 0.028 S 0.68 0.031 S 363 19879 CGPG4009 SENSE 2.117 0.029 S 0.16 0.139 T 0.091 0.202 / 0.545 0.028 S 347 72715 CGPG5521 SENSE 1.078 0.002 S 0.207 0.141 T 0.176 0.014 S 0.519 0.015 S 407 72771 CGPG2166 SENSE 0.564 0.256 / −0.019 0.857 / 0.206 0.013 S 0.306 0.05 S 440 72903 CGPG5584 SENSE 0.645 0.196 T −0.031 0.569 / 0.097 0.389 / 0.482 0.015 S 411 73054 CGPG5754 SENSE 1.52 0.035 S 0.198 0.089 T 0.098 0.187 T 0.696 0.004 S 476 74107 CGPG6590 SENSE 0.772 0.036 S 0.411 0 S 0.431 0.001 S 1.579 0 S 478 74131 CGPG6592 SENSE 1.747 0.044 S 0.316 0.004 S 0.115 0.01 S 0.663 0.001 S 442 74587 CGPG6774 SENSE 1.754 0.01 S 0.121 0.243 / 0.194 0.001 S 0.681 0 S S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2 /: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset

E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.

T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, ½×MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.

Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1L. Table 8 provides a list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants.

TABLE 8 Seedling Root Length Root Length Weight Pep Growth Stage at day 11 at day 14 at day 14 SEQ Con- Orienta- RS p- p- p- p- ID Gene struct_id tion mean value c delta value c delta value c delta value c 413 19707 CGPG4179 SENSE 2.653 0.019 S 0.074 0.58 T −0.031 0.81 / 0.427 0.063 T 414 19951 CGPG3941 SENSE 1.432 0.134 T 0.017 0.864 T −0.08 0.28 / 0.476 0.054 T 415 19967 CGPG4032 SENSE 2.691 0.014 S 0.01 0.934 T 0.19 0.039 S 0.537 0.056 T 416 70543 CGPG3815 SENSE 1.735 0.077 T 0.084 0.561 T 0.323 0.007 S 0.676 0.006 S 402 70681 CGPG4584 SENSE 2.528 0.006 S −0.065 0.682 / −0.179 0.05 / 0.374 0.146 T 417 70707 CGPG1273 ANTI- 1.42 0.095 T 0.248 0.006 S 0.3 0.002 S 0.331 0.007 S SENSE 418 70719 CGPG1712 ANTI- 1.43 0.106 T −0.007 0.968 / 0.101 0.255 T 0.155 0.349 T SENSE 419 71134 CGPG817 SENSE 1.478 0.13 T 0.119 0.15 T 0.035 0.684 T 0.198 0.277 T 420 71146 CGPG2928 SENSE 2.624 0.016 S 0.227 0.101 T 0.242 0.1 T 0.278 0.296 T 405 71508 CGPG1541 SENSE 3.153 0.001 S 0.446 0.054 T 0.384 0.048 S 0.782 0.003 S 421 71660 CGPG4690 SENSE 2.893 0.005 S −0.024 0.795 / 0.225 0.074 T 0.165 0.329 T 403 71663 CGPG4638 SENSE 0.116 0.444 T −0.061 0.536 / 0.358 0.041 S 0.058 0.702 T 439 71928 CGPG1617 SENSE 2.076 0.041 S 0.353 0.013 S 0.289 0.035 S 0.531 0.035 S 318 72081 CGPG5279 SENSE 1.262 0.138 T 0.174 0.243 T 0.2 0.197 T −0.105 0.714 / 408 72085 CGPG5228 SENSE 4 0 S 0.22 0.046 S 0.371 0.004 S 0.929 0 S 422 72086 CGPG5236 SENSE 2.589 0.022 S 0.281 0.033 S 0.136 0.314 T 0.32 0.043 S 423 72632 CGPG4852 SENSE 1.663 0.053 T 0.254 0.069 T 0.094 0.548 T 0.471 0.015 S 424 72716 CGPG5529 SENSE 2.914 0.004 S 0.146 0.058 T 0.007 0.925 T 0.582 0.016 S 425 72723 CGPG1848 SENSE 2.138 0.066 T 0.043 0.728 T 0.2 0.068 T −0.326 0.225 / 409 72744 CGPG5563 SENSE 1.636 0.05 / 0.195 0.151 T 0.059 0.751 T 0.539 0.005 S 404 72769 CGPG5573 SENSE 2.207 0.055 T 0.086 0.464 T 0.134 0.162 / 0.389 0.086 T 407 72771 CGPG2166 SENSE 2.569 0.021 S 0.169 0.221 T 0.192 0.038 S 0.56 0.018 S 440 72903 CGPG5584 SENSE 2.161 0.061 T 0.035 0.856 T 0.245 0.162 T 0.06 0.871 T 510 72921 CGPG5781 SENSE 2.249 0.025 S 0.034 0.788 T 0.225 0.077 T 0.306 0.28 T 391 72948 CGPG5617 SENSE 2.267 0.005 S 0.054 0.495 T 0.117 0.019 S 0.289 0.077 T 445 72961 CGPG5591 SENSE 1.19 0.14 T 0.037 0.76 T 0.136 0.339 T 0.588 0.027 S 511 72968 CGPG5772 SENSE 3.142 0.007 S 0.178 0.072 T −0.031 0.781 / 0.698 0.016 S 426 72987 CGPG1787 SENSE 2.055 0.078 T −0.058 0.64 / 0.097 0.144 T 0.325 0.054 T 438 72994 CGPG5803 SENSE 2.674 0.013 S 0.245 0.124 T 0.07 0.657 T 0.599 0.035 S 441 73017 CGPG5733 SENSE 4 0 S 0.343 0.09 T 0.462 0.002 S 0.996 0 S 410 73039 CGPG810 SENSE 4 0 S 0.319 0.019 S 0.237 0.05 / 0.426 0.011 S 411 73054 CGPG5754 SENSE 3.048 0.002 S 0.556 0.003 S 0.26 0.063 T 1.12 0.002 S 446 73070 CGPG5627 SENSE 3.439 0.001 S 0.139 0.49 T 0.19 0.135 T 0.24 0.365 T 447 73475 CGPG6385 SENSE 1.933 0.033 S 0.04 0.476 T 0.009 0.897 T 0.459 0.073 T 412 73501 CGPG6456 SENSE 1.29 0.121 T 0.143 0.118 T 0.043 0.564 T 0.433 0.013 S 427 74109 CGPG6606 SENSE 3.517 0 S 0.159 0.136 T 0.249 0.004 S 0.333 0.025 S 428 74140 CGPG6569 SENSE 2.05 0.039 S 0.168 0.138 T 0.196 0.086 T 0.71 0.012 S 429 74191 CGPG6597 SENSE 2.565 0.019 S 0.336 0.092 T 0.199 0.112 T 0.54 0.02 S 406 74248 CGPG5476 SENSE 3.158 0.007 S 0.14 0.192 T 0.204 0.051 T 0.377 0.037 S 430 74265 CGPG5356 SENSE 2.208 0.023 S 0.317 0.034 S 0.419 0.006 S 0.577 0.008 S 431 74369 CGPG6076 SENSE 3.522 0 S 0.347 0.045 S 0.272 0.107 T 0.624 0.02 S 442 74587 CGPG6774 SENSE 3.325 0.002 S 0.073 0.468 T 0.414 0.002 S 0.577 0.016 S 405 71508 CGPG1541 SENSE 3.153 0.001 S 0.446 0.054 T 0.384 0.048 S 0.782 0.003 S 439 71928 CGPG1617 SENSE 2.076 0.041 S 0.353 0.013 S 0.289 0.035 S 0.531 0.035 S 422 72086 CGPG5236 SENSE 2.589 0.022 S 0.281 0.033 S 0.136 0.314 / 0.32 0.043 S 469 72455 CGPG4742 SENSE 1.367 0.15 T 0.026 0.764 / 0.024 0.786 / 0.255 0.04 S 382 72466 CGPG4767 SENSE 0.735 0.101 T 0.068 0.333 / 0.249 0.006 S −0.341 0.278 / 373 72633 CGPG4853 SENSE 1.179 0.122 T 0.227 0.009 S 0.097 0.065 T 0.442 0.013 S 370 72757 CGPG5572 SENSE 2.272 0.017 S 0.125 0.361 / 0.11 0.317 / 0.425 0.047 S 472 72992 CGPG5777 SENSE 2.233 0.056 T 0.176 0.261 / 0.116 0.34 / 0.511 0.019 S 438 72994 CGPG5803 SENSE 2.674 0.013 S 0.245 0.124 T 0.07 0.657 / 0.599 0.035 S 441 73017 CGPG5733 SENSE 4 0 S 0.343 0.09 T 0.462 0.002 S 0.996 0 S 411 73054 CGPG5754 SENSE 3.048 0.002 S 0.556 0.003 S 0.26 0.063 T 1.12 0.002 S 300 73507 CGPG6504 SENSE 0.343 0.334 / 0.347 0.007 S 0.261 0.032 S 0.3 0.122 T 352 73515 CGPG6473 SENSE 3.336 0.002 S 0.279 0.009 S 0.241 0.003 S 0.328 0.05 S 428 74140 CGPG6569 SENSE 2.05 0.039 S 0.168 0.138 T 0.196 0.086 T 0.71 0.012 S 430 74265 CGPG5356 SENSE 2.208 0.023 S 0.317 0.034 S 0.419 0.006 S 0.577 0.008 S 431 74369 CGPG6076 SENSE 3.522 0 S 0.347 0.045 S 0.272 0.107 T 0.624 0.02 S 422 74587 CGPG6774 SENSE 3.325 0.002 S 0.073 0.468 / 0.414 0.002 S 0.577 0.016 S S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2 /: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset

F. Cold Shock Tolerance Screen

This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.

Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing ½× Gamborg Salts with 0.8 Phytagel™, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. Table 9 provides a list of recombinant nucleotides that improve cold shock stress tolerance in plants.

TABLE 9 difference in rosette area rosette area rosette area between day 28 Pep at day 8 at day 28 and day 8 SEQ Con- Orienta- p- p- p- ID struct_id Gene tion delta value c delta value c delta value c 270 14324 CGPG1560 SENSE 0.054 0.429 / 0.258 0.017 S −0.071 0.631 / 271 17484 CGPG2630 SENSE −0.189 0.759 / 0.544 0.025 S 0.275 0.121 T 272 19109 CGPG1381 ANTI- −0.016 0.523 / 0.541 0.008 S 0.818 0.014 S SENSE 273 70423 CGPG3165 SENSE 0.316 0.012 S 0.521 0.022 S 0.89 0.018 S 274 70424 CGPG3180 SENSE 0.474 0.003 S 0.695 0.003 S 0.693 0.043 S 275 70480 CGPG3833 SENSE −0.066 0.591 / 0.175 0.159 T 0.474 0.059 T 276 70509 CGPG2420 SENSE 0.023 0.438 / 0.117 0.216 / 0.609 0.032 S 277 70647 CGPG4334 SENSE −0.508 0.894 / 0.604 0.049 S 0.895 0.047 S 278 70675 CGPG4519 SENSE 0.2 0.2 / 0.303 0.153 T 0.507 0.034 S 279 70829 CGPG518 SENSE −0.319 0.823 / 0.804 0.002 S 1.082 0.002 S 280 70849 CGPG596 SENSE −0.039 0.564 / 0.698 0.001 S 0.707 0.001 S 281 71627 CGPG1270 SENSE −0.146 0.748 / 0.349 0.05 T 0.3 0.12 T 282 71934 CGPG2294 SENSE −0.068 0.796 / 0.757 0 S 0.922 0 S 283 72615 CGPG4829 SENSE 0.477 0.007 S 0.834 0 S 0.979 0.001 S 286 73559 CGPG6535 SENSE 0.143 0.093 T −0.265 0.878 / −0.344 0.821 / 287 74251 CGPG5489 SENSE 0.377 0.021 S 0.439 0.045 S 0.45 0.07 T 389 70437 CGPG3706 SENSE −0.273 0.916 / 0.147 0.165 T 0.682 0.034 S 402 70681 CGPG4584 SENSE 0.352 0.155 T 0.252 0.261 / 0.269 0.328 / 403 71663 CGPG4638 SENSE 0.358 0.013 S 0.032 0.423 / −0.031 0.585 / 404 72769 CGPG5573 SENSE 0.381 0.049 S 0.881 0.006 S 1.102 0.005 S 407 72771 CGPG2166 SENSE 0.993 0 S 1.381 0.003 S 1.536 0.003 S 432 70217 CGPG6 SENSE 0.275 0.067 T 0.126 0.289 / 0.362 0.215 / 433 72711 CGPG1846 SENSE 0.774 0.001 S 0.579 0.004 S 0.429 0.038 S 438 72994 CGPG5803 SENSE 0.116 0.381 / 0.708 0.068 T 0.744 0.069 T 510 72921 CGPG5781 SENSE 0.265 0.057 T 0.31 0.162 T 0.367 0.11 T 414 19951 CGPG3941 SENSE 0.729 0.006 S 0.473 0.017 S 0.846 0.006 S 273 70423 CGPG3165 SENSE 0.316 0.012 S 0.521 0.022 S 0.89 0.018 S 416 70543 CGPG3815 SENSE 1.584 0 S 0.86 0 S 0.82 0.002 S 368 71617 CGPG1227 SENSE 0.204 0.136 T 0.408 0.025 S 0.458 0.057 T 439 71928 CGPG1617 SENSE 0.104 0.265 / 0.786 0 S 0.836 0.001 S 382 72466 CGPG4767 SENSE 0.497 0.017 S 0.565 0.017 S 0.963 0.002 S 383 72524 CGPG4770 SENSE 0.438 0.02 S 0.377 0.025 S 0.385 0.043 S 409 72744 CGPG5563 SENSE 0.52 0.058 T 0.859 0.026 S 0.454 0.189 T 444 72967 CGPG5742 SENSE 0.955 0 S 0.629 0.009 S 0.403 0.189 T 435 73518 CGPG6497 SENSE 0.114 0.278 / 0.319 0.01 S 0.195 0.114 T 306 74136 CGPG6632 SENSE 0.606 0.007 S 0.523 0.036 S 0.598 0.025 S 398 74240 CGPG5454 SENSE −0.099 0.644 / 1.277 0.003 S 1.498 0.006 S 431 74369 CGPG6076 SENSE 0.623 0.002 S 0.62 0.04 S 0.737 0.096 T S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2 /: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset.

G. Cold Germination Tolerance Screen

This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.

T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of ½× Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% Phytagel™ (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m²/s. At 28 days post planting, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.

The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1L. Table 10 provides a list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants.

TABLE 10 Growth stage Root Length Pep at day 28 at day 28 SEQ Con- Orienta- RS p- p- ID struct_id Gene tion mean value c delta value c 288 19631 CGPG3627 SENSE 2.229 0.052 T 0.094 0.252 / 289 70121 CGPG2380 SENSE 2.732 0.042 S 0.126 0.238 / 290 70654 CGPG4352 SENSE 2.474 0.026 S 0.263 0.019 S 291 70696 CGPG4590 SENSE 3.092 0.01 S 0.086 0.145 T 292 70713 CGPG1462 ANTI- 4 0 S 0.268 0.014 S SENSE 293 70740 CGPG3700 SENSE 2.485 0.024 S 0.244 0.012 S 294 71321 CGPG4418 SENSE 1.837 0.126 T 0.014 0.474 / 295 71835 CGPG4634 SENSE 3.349 0.002 S 0.353 0.036 S 296 72934 CGPG5798 SENSE 2.222 0.023 S 0.243 0.101 T 297 72945 CGPG5787 SENSE 3.478 0.001 S 0.236 0.011 S 298 72980 CGPG5773 SENSE 3.265 0.003 S 0.239 0.006 S 299 73504 CGPG6480 SENSE 4 0 S 0.521 0 S 300 73507 CGPG6504 SENSE 4 0 S 0.404 0.001 S 301 73573 CGPG6462 SENSE 4 0 S 0.268 0.004 S 302 73586 CGPG6471 SENSE 4 0 S 0.314 0.064 T 303 73770 CGPG5435 SENSE 3.091 0.01 S 0.303 0.085 T 304 74105 CGPG6574 SENSE 3.329 0.002 S 0.109 0.116 T 305 74111 CGPG6622 SENSE 2.226 0.021 S 0.445 0.006 S 306 74136 CGPG6632 SENSE 3.192 0.005 S 0.328 0.002 S 307 74139 CGPG6561 SENSE 4 0 S 0.254 0.092 T 308 74267 CGPG5364 SENSE 3.054 0.002 S 0.3 0 S 309 74291 CGPG5363 SENSE 4 0 S 0.142 0.142 T 310 74318 CGPG5826 SENSE 4 0 S 0.272 0.008 S 311 74319 CGPG5831 SENSE 3.207 0.005 S 0.201 0.002 S 312 74324 CGPG5885 SENSE 3.144 0.007 S 0.232 0.017 S 313 74512 CGPG32 SENSE 4 0 S 0.332 0.011 S 314 74583 CGPG6649 SENSE 3.249 0.004 S 0.28 0.001 S 315 70427 CGPG3067 SENSE 1.567 0.108 T 0.222 0.044 S 352 73515 CGPG6473 SENSE 4 0 S 0.324 0.003 S 353 74684 CGPG6360 SENSE 2.927 0.004 S 0.426 0.003 S 373 72633 CGPG4853 SENSE 2.121 0.027 S 0.289 0.048 S 405 71508 CGPG1541 SENSE 1.99 0.039 S 0.263 0.033 S 406 74248 CGPG5476 SENSE 2.385 0.011 S 0.217 0.017 S 434 70932 CGPG4089 SENSE 3.268 0.003 S 0.146 0.067 T 435 73518 CGPG6497 SENSE 3.373 0.002 S 0.352 0.032 S 439 71928 CGPG1617 SENSE 3.062 0.011 S 0.18 0.002 S 504 74259 CGPG5343 SENSE 3.511 0 S 0.308 0.024 S 505 72463 CGPG4760 SENSE 2.736 0.01 S 0.032 0.386 / 506 72902 CGPG5597 SENSE 3.105 0.009 S 0.278 0.037 S 507 74572 CGPG6640 SENSE 4 0 S 0.125 0.155 T 508 73055 CGPG5768 SENSE 3.173 0.006 S 0.407 0.004 S 413 19707 CGPG4179 SENSE 1.829 0.061 T 0.169 0.018 S 360 19779 CGPG4113 SENSE 4 0 S 0.213 0.017 S 361 19833 CGPG4074 SENSE / / / 0.292 0.022 S 363 19879 CGPG4009 SENSE 4 0 S 0.34 0.001 S 514 19980 CGPG3914 SENSE 0.798 0.122 T 0.278 0.011 S 273 70423 CGPG3165 SENSE 2.906 0.004 S 0.105 0.114 T 459 70725 CGPG2097 ANTI- 1.949 0.044 S 0.122 0.148 T SENSE 461 71112 CGPG934 SENSE 2.579 0.018 S 0.185 0.17 T 444 72967 CGPG5742 SENSE 4 0 S 0.287 0.007 S 438 72994 CGPG5803 SENSE 1.072 0.098 T 0.161 0.04 S 521 73488 CGPG6394 SENSE 4 0 S 0.211 0.012 S 477 74117 CGPG6575 SENSE 2.567 0.02 S 0.123 0.04 S S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2 /: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined compared to the reference in the current dataset

H. Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes may confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response are associated with higher yields. Genes that favor growth under low light conditions may also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low. light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.

T2 seeds were plated on glufosinate selection plates with ½ MS medium. Seeds were sown on ½×MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m²/s, far/red ratio (655/665/725/735) ˜0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1L.

Table 11 provides a list of recombinant DNA constructs that improve shade tolerance in plants

TABLE 11 flowerbud Number of seedling formation Leaf Angle Petiole length rosette leaves weight Pep at day 23 at day 23 at day 23 at day 23 at day 23 SEQ Con- Orienta- RS p- RS RS p- RS p- p- ID struct_id tion mean value c mean p-value c mean value c mean value c delta value c 376 70426 SENSE 0.719 0.09 T 0.163 0.244 / −0.206 0.185 T −0.225 0.984 / 0.484 0.003 S 377 70772 SENSE −0.501 0.858 / −0.066 0.724 / −0.692 0.004 S −0.626 0.983 / −0.693 0.023 / 378 71137 SENSE 0.26 0.384 / 0.11 0.168 T −0.869 0.029 S 1.064 0.073 T −0.453 0.288 / 379 71529 SENSE 0.89 0.108 T −0.014 1 / −0.44 0.006 S 1.817 0.024 S −0.022 0.914 / 380 71601 SENSE 1.79 0.066 T 0.195 0.172 T −0.057 0.59 / −0.483 0.961 / 0.264 0.308 / 381 72362 SENSE 1.763 0.072 T 0.218 0.262 / −0.109 0.242 / 0.003 0.484 / 0.311 0.119 T 374 72456 SENSE 0.563 0.313 / 0.751 0.186 T −0.848 0.001 S −0.624 0.999 / −0.85 0.025 / 382 72466 SENSE −1.382 1 / −0.092 0.645 / −0.894 0 S 1.212 0.122 T −0.879 0.027 / 383 72524 SENSE −0.153 1 / 0.152 0.09 T −0.952 0.002 S −0.57 1 / −1.269 0.01 / 373 72633 SENSE 3.513 0 S −0.431 0.977 / −0.426 0.001 S −0.735 0.994 / 0.269 0.129 T 375 72963 SENSE −0.911 0.988 / 0.236 0.396 / −0.753 0.003 S −0.083 0.665 / −0.485 0.015 / 384 73085 SENSE −0.073 0.756 / 1.212 0.122 T 0.175 0.097 / 4 0 S 0.51 0.027 S 385 74241 SENSE −0.195 0.935 / −0.098 0.64 / −1.077 0.01 S −0.821 0.999 / −1.024 0.002 / 386 74247 SENSE 0.43 0.16 T 0.203 0.095 T −0.22 0.197 T 1.166 0.07 T 0.483 0.018 S 387 74284 SENSE 0.062 0.22 / 2.077 0.038 S −0.014 0.943 / 2.348 0.04 S 0.03 0.938 / 388 74652 SENSE 0.015 0.442 / 0.093 0.455 / −0.073 0.621 / 0.967 0.184 T 0.173 0.569 / 363 19879 SENSE 0.585 / / 0 / / 0.152 / / 0 / / 0.708 / / 293 70740 SENSE 0.44 0.137 T 0.609 0.103 T 0.161 0.186 / / / / 0.45 0.001 S 316 71811 SENSE −0.025 0.637 / 0.725 0.113 T −0.205 0.01 S / / / −0.517 0.037 / 397 71840 SENSE 1.498 0.12 T 0.712 0.178 T −0.379 0.001 S −0.188 0.765 / −0.95 0.124 / 468 72450 SENSE 0.068 0.349 / −0.042 1 / 0.166 0.016 / 1.62 0.049 S 0.501 0.038 S 370 72757 SENSE 3.595 0 S 0.872 0.079 T 0.064 0.138 / −0.767 0.997 / 0.546 0.001 S 444 72967 SENSE 1.829 0.063 T 1.123 0.065 T 0.133 0.196 / −0.18 0.794 / 0.509 0.008 S 511 72968 SENSE 0.185 0.005 S 0.48 0.179 T 0.22 0.162 / 1.698 0.085 T 0.382 0.031 S 300 73507 SENSE 0.248 0.086 T 0.307 0.287 / −0.304 0.031 S / / / 0.05 0.86 / 307 74139 SENSE −0.011 0.556 / −0.071 1 / −0.124 0.068 T −0.511 1 / −0.413 0.124 / 535 74474 SENSE 0.755 0.154 T −0.145 0.857 / 0.142 0.396 / / / / 0.765 0.005 S 400 74610 SENSE 0.572 0.222 / 0.177 0.22 / −0.18 0.071 T / / / −0.341 0.173 / S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2 /: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined compared to the reference in the current dataset.

I. Early Plant Growth and Development Screen

This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.

T2 seeds were plated on glufosinate selection plates and grown under standard conditions (˜100 □E/m²/s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.

Table 12 provides a list recombinant DNA constructs that improve early plant growth and development.

TABLE 12 Seedling Pep Root Length Weight SEQ Con- Orienta- p- p- ID struct_id gene tion delta value c delta value c 432 70217 CGPG6 SENSE 0.038 0.469 / 0.375 0.047 S 433 72711 CGPG1846 SENSE 0.132 0.021 S 0.601 0.001 S 434 70932 CGPG4089 SENSE 0.328 0.005 S 0.473 0.017 S 435 73518 CGPG6497 SENSE 0.287 0 S 0.634 0.036 S 436 19771 CGPG4011 SENSE 0.218 0.076 T 0.581 0.018 S 437 73549 CGPG6460 SENSE 0.139 0.003 S 0.349 0.03 S 438 72994 CGPG5803 SENSE 0.44 0.004 S 0.791 0.001 S 439 71928 CGPG1617 SENSE 0.073 0.427 / 0.494 0.005 S 440 72903 CGPG5584 SENSE 0.298 0.002 S 0.399 0.044 S 441 73017 CGPG5733 SENSE 0.284 0.004 S 0.199 0.488 / 442 74587 CGPG6774 SENSE 0.111 0.075 T 0.538 0.006 S 443 72453 CGPG4735 SENSE 0.215 0.005 S 0.416 0.069 T 444 72967 CGPG5742 SENSE 0.103 0.212 / 0.568 0.008 S 445 72961 CGPG5591 SENSE 0.177 0.046 S 0.548 0.006 S 446 73070 CGPG5627 SENSE 0.221 0.007 S 0.652 0.005 S 447 73475 CGPG6385 SENSE 0.084 0.014 S 0.336 0.014 S 448 72916 CGPG1814 SENSE 0.182 0.067 T 0.353 0.012 S 449 72969 CGPG5789 SENSE 0.133 0.245 / 0.45 0.069 T 450 74449 CGPG6659 SENSE 0.314 0.002 S 0.579 0.027 S 451 16615 CGPG2539 SENSE 0.223 0.023 S 0.571 0.009 S 452 19187 CGPG3310 SENSE 0.264 0.001 S 0.51 0.08 T 453 19648 CGPG3134 SENSE 0.218 0.013 S 0.27 0.106 T 454 70354 CGPG3995 SENSE 0.152 0.046 S 0.406 0.029 S 455 70421 CGPG2942 SENSE 0.179 0.225 / 0.581 0.013 S 456 70459 CGPG3758 SENSE 0.187 0.036 S −0.034 0.884 / 457 70465 CGPG3775 SENSE −0.009 0.899 / 0.188 0.196 T 458 70683 CGPG4587 SENSE 0.133 0.088 T 0.402 0.014 S 459 70725 CGPG2097 ANTI- 0.326 0.001 S 0.116 0.548 / SENSE 460 70852 CGPG1465 SENSE 0.237 0 S 0.297 0.127 T 461 71112 CGPG934 SENSE 0.199 0.013 S 0.316 0.034 S 462 71127 CGPG945 SENSE 0.097 0.02 S 0.4 0.054 T 463 71132 CGPG1561 SENSE 0.195 0.02 S 0.08 0.524 / 464 71217 CGPG95 SENSE 0.234 0 S 0.566 0.036 S 465 71645 CGPG4688 SENSE 0.475 0.003 S 0.361 0.133 T 466 71726 CGPG3894 SENSE 0.223 0.056 T 0.458 0.033 S 467 72432 CGPG4562 SENSE 0.209 0 S 0.581 0.041 S 468 72450 CGPG4732 SENSE 0.335 0 S 0.79 0 S 469 72455 CGPG4742 SENSE 0.278 0.019 S 0.482 0.051 T 470 72727 CGPG5522 SENSE 0.123 0.002 S 0.315 0.004 S 471 72817 CGPG4987 SENSE 0.254 0.023 S 0.485 0 S 472 72992 CGPG5777 SENSE 0.219 0.023 S 0.664 0.015 S 473 73007 CGPG5760 SENSE 0.139 0.093 T 0.462 0.008 S 474 73073 CGPG5688 SENSE 0.164 0.022 S 0.285 0.247 / 475 73506 CGPG6496 SENSE 0.512 0 S 0.986 0 S 476 74107 CGPG6590 SENSE 0.282 0.002 S 0.538 0.057 T 477 74117 CGPG6575 SENSE 0.211 0.002 S 0.449 0.005 S 478 74131 CGPG6592 SENSE 0.142 0.047 S 0.586 0.003 S 479 74344 CGPG5929 SENSE 0.27 0.01 S 0.474 0.105 T 323 13502 CGPG1354 SENSE 0.57 0.002 S 0.625 0 S 330 18259 CGPG3368 SENSE 0.226 0.026 S 0.551 0.021 S 361 19833 CGPG4074 SENSE 0.259 0 S 0.472 0.016 S 334 70417 CGPG3427 SENSE 0.187 0.056 T 0.113 0.65 / 273 70423 CGPG3165 SENSE 0.03 0.747 / 0.185 0.007 S 515 70435 CGPG3701 SENSE 0.131 0.051 T 0.394 0.014 S 493 70601 CGPG2917 SENSE 0.177 0 S 0.365 0.063 T 291 70696 CGPG4590 SENSE 0.079 0.205 / 0.331 0.024 S 365 70738 CGPG3195 SENSE 0.012 0.864 / 0.472 0.003 S 293 70740 CGPG3700 SENSE 0.103 0.082 T 0.387 0.021 S 419 71134 CGPG817 SENSE 0.063 0.548 / 0.279 0.066 T 422 72086 CGPG5236 SENSE 0.083 0.234 / 0.398 0.05 S 505 72463 CGPG4760 SENSE −0.131 0.416 / 0.422 0.003 S 285 73014 CGPG5692 SENSE 0.11 0.391 / 0.373 0.096 T 521 73488 CGPG6394 SENSE 0.018 0.793 / 0.398 0.1 T 299 73504 CGPG6480 SENSE −0.152 0.479 / 0.538 0.012 S 300 73507 CGPG6504 SENSE 0.15 0.002 S 0.053 0.854 / 301 73573 CGPG6462 SENSE 0.112 0.07 T 0.375 0.006 S 302 73586 CGPG6471 SENSE 0.309 0 S 0.611 0 S 303 73770 CGPG5435 SENSE 0.21 0.059 T 0.281 0.484 / 509 74103 CGPG6558 SENSE 0.374 0 S 0.561 0.024 S 428 74140 CGPG6569 SENSE 0.16 0.017 S 0.376 0.066 T 527 74262 CGPG5353 SENSE 0.203 0.012 S 0.375 0.045 S 430 74265 CGPG5356 SENSE 0.107 0.259 T 0.432 0.067 T 529 74302 CGPG5384 SENSE 0.115 0.101 T 0.269 0.056 T 431 74369 CGPG6076 SENSE 0.138 0.03 S 0.195 0.21 / 534 74465 CGPG6692 SENSE 0.2 0.02 S 0.688 0.042 S 507 74572 CGPG6640 SENSE 0.162 0.023 S 0.617 0 S 314 74583 CGPG6649 SENSE 0.144 0.023 S 0.403 0.008 S S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2 /: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset

J. Late Plant Growth and Development Screen

This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants. Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, 1-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft³. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of ˜5 per 2½″ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of ˜200 μmoles/m²/s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.

Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling ˜3 days after the selection was applied.

The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.

Table 13 provides a list of recombinant DNA constructs that improve late plant growth and development.

TABLE 13 Rosette Dry Rosette Seed Dry Silique Dry Slilique Pep Weight Radius Weight Weight Length SEQ Con- Orienta- p- p- p- p- p- ID struct_id tion delta value c delta value c delta value c delta value c delta value c 480 14320 SENSE −0.145 0.94 / 0.137 0.038 S −0.702 1 / 0.477 0.002 S 0.016 0.276 / 481 16756 SENSE 0.485 0.016 S 0.223 0.025 S 0.148 0.042 S 0.481 0.002 S 0.147 0.013 S 482 17448 SENSE −0.288 0.991 / −0.054 0.829 / 0.376 0.008 S 0.185 0.034 S −0.064 0.981 / 483 17633 SENSE 0.018 0.359 / 0.13 0.106 T 0.416 0.055 T 0.399 0.044 S 0.116 0.08 T 484 18876 SENSE 0.257 0.016 S 0.026 0.448 / −0.213 0.786 / 0.386 0 S −0.015 0.705 / 485 19120 ANTI- −0.252 0.936 / 0.022 0.006 S −1.042 0.95 / 0.165 0.076 T 0.046 0.021 S SENSE 486 19221 SENSE −0.316 0.986 / 0.153 0.097 T −0.35 0.903 / 0.394 0.068 T 0.183 0.028 S 487 70206 SENSE 0.125 0 S 0.074 0.18 T 0.712 0.022 S 0.12 0.026 S 0.019 0.403 / 488 70223 SENSE 0.197 0.026 S 0.23 0.018 S −0.781 0.998 / 0.134 0.039 S −0.205 0.915 / 489 70347 SENSE 0.156 0.038 S −0.082 0.868 / −0.153 0.752 / 0.405 0.006 S 0.056 0.068 T 490 70406 SENSE −0.275 0.948 / −0.245 0.992 / 0.759 0.025 S −0.282 0.949 / −0.121 0.939 / 491 70469 SENSE 0.032 0.392 / 0.348 0.004 S −0.733 0.996 / 0.325 0.059 T −0.141 0.922 / 492 70564 SENSE 0.17 0.037 S 0.051 0.234 / 0.772 0 S −0.381 0.977 / −0.015 0.655 / 493 70601 SENSE 0.231 0.086 T 0.247 0.004 S −0.257 0.959 / 0.323 0.024 S 0.082 0.03 S 494 70612 SENSE 0.053 0.112 T 0.082 0.096 T 1.049 0.011 S −0.212 0.992 / 0.07 0.004 S 495 70720 ANTI- −0.16 0.898 / 0.028 0.384 / 1.312 0.009 S 0.219 0.121 T 0.128 0.018 S SENSE 496 70735 SENSE −0.058 0.672 / 0.156 0.087 T 0.421 0.036 S 0.532 0.003 S 0.033 0.038 S 497 70846 SENSE 0.086 0.255 / 0.134 0.063 T −0.33 0.918 / 0.142 0.151 T 0.011 0.439 / 498 70923 SENSE 0.484 0.011 S 0.108 0.081 T −0.4 0.844 / 0.091 0.198 T 0.099 0.001 S 499 71149 SENSE −1.085 0.993 / −0.043 0.681 / 0.346 0.017 S 0.136 0.133 T 0.066 0.077 T 500 71608 SENSE −0.849 0.907 / −0.132 0.976 / 0.816 0.006 S 0.279 0.004 S 0.038 0.238 / 501 71739 SENSE −0.275 0.937 / −0.107 0.955 / 0.334 0 S −0.075 0.815 / −0.119 0.874 / 502 72014 SENSE −0.038 0.94 / 0.07 0.278 / 0.732 0.06 T −0.026 0.584 / −0.013 0.596 / 503 72051 SENSE 0.026 0.28 / 0.311 0.003 S 0.222 0.236 / 0.453 0.009 S 0.052 0.168 T 432 70217 SENSE −0.202 0.893 / 0.203 0.024 S −0.079 0.743 / 0.27 0.07 T −0.063 0.856 / 454 70354 SENSE 0.134 0.147 T −0.119 0.684 / 0.48 0.02 S −0.119 0.736 / 0.014 0.3 / 334 70417 SENSE −0.35 0.988 / −0.041 0.637 / 0.69 0.012 S −0.136 0.978 / 0.004 0.411 / 515 70435 SENSE 0.664 0.014 S 0.146 0.036 S 0.138 0.097 T 0.33 0.038 S 0.039 0.16 T 457 70465 SENSE −0.106 0.79 / / / / 0.883 0.001 S −0.27 0.945 / −0.097 0.818 / 460 70852 SENSE 0.178 0.031 S 0.145 0.034 S −0.525 0.904 / −0.315 0.861 / −0.059 0.774 / 405 71508 SENSE 0.195 0.162 T 0.251 0.017 S −0.515 0.929 / −0.322 0.997 / −0.196 0.982 / 295 71835 SENSE 0.139 0.055 T 0.163 0.049 S 0.538 0.019 S 0.27 0.068 T 0 0.496 / 467 72432 SENSE 0.146 0.021 S 0.139 0.02 S 0.325 0.012 S 0.074 0.149 T −0.106 0.879 / 443 72453 SENSE 0.204 0.037 S 0.116 0.046 S −2.198 0.995 / 0.016 0.448 / −0.013 0.534 / 433 72711 SENSE 0.292 0.058 T 0.143 0.024 S −0.114 0.76 / −0.04 0.667 / −0.093 0.946 / 449 72969 SENSE 0.046 0.072 T −0.158 0.887 / 0.39 0.031 S 0.477 0.001 S 0.095 0.054 T 426 72987 SENSE 0.385 0.006 S / / / 0.098 0.104 T 0.153 0.016 S 0.057 0.061 T 525 74022 SENSE 0.11 0.226 / −0.069 0.844 / 0.71 0.009 S −0.05 0.613 / −0.004 0.544 / 287 74251 SENSE −0.56 0.961 / −0.184 0.916 / 0.611 0.001 S 0.174 0.229 / −0.137 0.923 / 537 74507 SENSE 0.255 0.017 S 0.178 0.03 S 0.318 0.032 S 0.188 0.011 S 0.013 0.078 T 313 74512 SENSE −0.247 0.953 / 0.107 0.015 S −0.073 0.86 / 0.113 0.034 S 0.05 0.175 T S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents data points not determined or the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2 /: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset

K. Limited Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.

T2 seeds were plated on glufosinate selection plates containing 0.5× N-Free Hoagland's T 0.1 mM NH₄NO₃ T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i.e., viable or non-viable) and root length. After 21 days of growth, plants were scored for visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.

The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1L.

Table 14 provides a list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants.

TABLE 14 Number of Flowerbud Number of rosette Seedling Pep formation green leaves Root Length leaves Weight SEQ Con- Orienta- RS p- RS p- p- RS p- ID struct_id tion mean value c mean value c delta value c mean p-value c delta value c 375 72963 SENSE 1.1 0.004 S 0.293 0.021 S −0.446 0.002 S −0.246 0.786 / 0.137 0.001 S 389 70437 SENSE −0.28 0.982 / −0.08 0.769 / 0.259 0.006 S 0.5 0.005 S 0.133 0.003 S 390 71633 SENSE 0.26 0.26 / 0.254 0.114 T −0.1 0.539 / 0.647 0.06 T 0.106 0.023 S 391 72948 SENSE 0.587 0.06 T 0.539 0.029 S −0.237 0.003 S 0.479 0.1 T 0.078 0.121 T 392 72519 SENSE 0.749 0.033 S 0.209 0.104 T −0.09 0.274 / 0.276 0.264 / 0.116 0.006 S 393 10475 SENSE 1.256 0.026 S 0.588 0.005 S −0.378 0.002 S 0.081 0.319 / 0.018 0.75 / 394 11120 ANTI- 0.795 0.033 S 0.608 0.015 S −0.45 0.001 S 0.287 0.106 T −0.041 0.387 / SENSE 395 19736 SENSE −0.24 0.907 / 0.355 0.033 S 0.014 0.864 / 0.64 0.006 S −0.075 0.005 / 396 71606 SENSE 0.605 0.088 T 0.176 0.11 T −0.033 0.708 / 1.239 0.005 S 0.133 0.002 S 397 71840 SENSE 0.408 0.235 / 0.879 0.006 S −0.137 0.248 / 0.524 0.032 S 0.066 0.198 T 398 74240 SENSE −0.06 0.602 / 0.13 0.076 T −0.107 0.306 / 0.714 0.034 S 0.108 0.002 S 399 74331 SENSE −0.44 1 / 0.054 0.203 / 0.132 0.055 T 1.045 0.021 S 0.134 0.003 S 400 74610 SENSE −0.59 1 / −0.08 0.922 / 0.289 0 S 1.241 0.017 S 0.137 0.001 S 401 75527 SENSE 0.242 0.228 / 0.376 0.028 S −0.183 0.045 / 0.352 0.083 T −0.005 0.8 / 316 71811 SENSE −0.45 0.91 / / / / −0.112 0.202 / 0.438 0.014 S −0.054 0.161 / 505 72463 SENSE −0.16 0.976 / / / / 0.112 0.109 T 0.366 0.024 S 0.13 0.006 S 351 75701 SENSE 0.736 0.048 S 0.07 0.861 / −0.4 0.018 S / / / −0.109 0.193 / S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05) T: represents the transgenic plants showed a trend of trait improvement compared than the reference with p < 0.2 /: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset

L. Statistic Analysis for Qualitative Responses

Table 15 provides a list of responses that were analyzed as qualitative responses

TABLE 15 response Screen categories (success vs. failure) wilting response Risk Soil drought tolerance screen non-wilted vs. wilted Score growth stage at day 14 heat stress tolerance screen 50% of plants reach stage 1.03 vs. not growth stage at day 14 salt stress tolerance screen 50% of plants reach stage 1.03 vs. not growth stage at day 14 PEG induced osmotic stress tolerance 50% of plants reach stage 1.03 vs. not screen growth stage at day 7 cold germination tolerance screen 50% of plants reach stage 0.5 vs. not number of rosette leaves Shade tolerance screen 5 leaves appeared vs. not at day 23 flower bud formation at Shade tolerance screen flower buds appear vs. not day 23 leaf angle at day 23 Shade tolerance screen >60 degree vs. <60 degree number of green leaves at limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not day 21 number of rosette leaves limited nitrogen tolerance screen 6 or 7 leaves appeared vs. not at day 21 Flower bud formation at limited nitrogen tolerance screen flower buds appear vs. not day 21

Plants were grouped into transgenic and reference groups and were scored as success or failure according to criteria in Table 15. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log₂ ^(RR). Subsequently the risk scores from multiple events for each transgene of interest were evaluated for statistical significance by t-test using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.

M. Statistic Analysis for Quantitative Responses

Table 16 provides a list of responses that were analyzed as quantitative responses.

TABLE 16 response screen seed yield Soil drought stress tolerance screen seedling weight at day 14 heat stress tolerance screen root length at day 14 heat stress tolerance screen seedling weight at day 14 salt stress tolerance screen root length at day 14 salt stress tolerance screen root length at day 11 salt stress tolerance screen seedling weight at day 14 PEG induced osmotic stress tolerance screen root length at day 11 PEG induced osmotic stress tolerance screen root length at day 14 PEG induced osmotic stress tolerance screen rosette area at day 8 cold shock tolerance screen rosette area at day 28 cold shock tolerance screen difference in rosette area cold shock tolerance screen from day 8 to day 28 root length at day 28 cold germination tolerance screen seedling weight at day 23 Shade tolerance screen petiole length at day 23 Shade tolerance screen root length at day 14 Early plant growth and development screen Seedling weight at day 14 Early plant growth and development screen Rosette dry weight at Late plant growth and development screen day 53 rosette radius at day 25 Late plant growth and development screen seed dry weight at day 58 Late plant growth and development screen silique dry weight at day 53 Late plant growth and development screen silique length at day 40 Late plant growth and development screen Seedling weight at day 21 Limited nitrogen tolerance screen Root length at day 21 Limited nitrogen tolerance screen

The measurements (M) of each plant were transformed by log₂ calculation. The Delta was calculated as log₂M(transgenic)−log₂M(reference). Subsequently the mean delta from multiple events of the transgene of interest was evaluated for statistical significance by t-test using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). The Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. The Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.

Example 2 Identification of Homologs

A BLAST searchable “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.

The All Protein Database was queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 240 through SEQ ID NO: 478 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.

The Organism Protein Database was queried using amino acid sequences of SEQ ID NO: 270 through SEQ ID NO: 538 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 539 to SEQ ID NO: 22568. The relationship of the homologs to the identified trait-improving genes on an amino acid sequence basis is found in Table 17 where the amino acid sequence of a protein encoded by a trait-improving DNA, e.g., SEQ ID NO:270, is followed by the amino acid sequences of protein encoded by homologous genes, e.g., SEQ ID NO:19844, 4248, 2761, 15944, etc. The source organism of each homolog is reported in the Sequence Listing.

TABLE 17 Sequence IDs for homolog proteins Seq ID NO: homolog Seq ID NOs 270: 19844 4248 2761 15944 11776 16144 10470 9742 6776 1010 2285 16333 9154 20620 16454 20025 8388 10646 1208 6001 1706 2448 14768 10226 12626 19846 9302 17295 17794 6354 5098 1789 6430 17749 821 10109 7542 17855 15562 17462 271: 1715 5418 7208 19338 7440 711 8113 17151 17592 18879 4807 8671 9936 11315 10681 3177 10519 6830 13563 12162 15155 1860 18072 20945 6715 15032 5192 10928 272: 0 273: 13771 8553 4219 10043 10800 8345 17501 13569 954 17197 6188 3760 13267 16169 8132 2667 11216 15637 4652 2270 10309 5708 18374 9446 12844 7790 7569 4786 9725 14187 12859 16948 18626 13741 5525 7877 4550 15544 9706 7616 14358 15163 13182 14560 16722 1129 1472 4261 10693 20144 6437 21413 17893 17984 17116 9925 19953 20648 983 2837 5663 2943 10465 1841 12497 6435 14763 13495 12676 7513 8363 16389 8162 7945 14956 15029 12433 22241 16071 13003 16940 18847 12354 7732 14013 5735 11505 8833 17658 16048 17609 575 6641 6331 3738 10842 18927 21518 20097 14117 5309 13744 15880 19484 9648 22509 1221 15515 6785 852 6466 17423 14164 658 8704 16710 19375 3306 14050 16883 16322 1722 15481 13636 10680 6347 3552 8885 21794 17703 22557 14777 21189 13711 3601 3968 3692 4003 20044 12943 19749 1865 6355 14902 6137 22370 19468 10410 21460 10451 17175 20965 12916 1206 6796 11329 9139 11008 10569 9058 7988 19743 20088 14111 8231 4522 18497 11952 2866 15466 3609 2403 16796 13539 14806 5364 12620 20699 12940 15426 4409 12452 5296 9156 629 12665 20947 3649 7530 274: 16167 5178 2412 10455 20036 11246 19666 6400 5573 22539 8547 21845 2413 20290 4036 19351 5886 6071 17184 9738 275: 14665 16694 12678 14928 21489 7918 1571 3959 2490 2517 14615 3788 10022 16096 21248 13293 8541 13446 6120 4360 3812 15574 18938 19203 2284 2215 10054 14052 9653 10183 17752 20776 4240 22343 8270 9192 17217 7374 12141 20657 7674 6445 2522 276: 12496 3460 13599 7043 9150 1664 277: 16949 9640 8150 2014 12188 5779 17876 14612 18293 11053 15958 15263 18370 20984 13094 18734 7380 10318 21641 12737 13028 20561 7087 10686 9894 7528 12573 16043 14846 20513 2802 8897 14716 10257 16407 2727 6151 1484 6831 16916 10146 17756 13193 7670 15946 7750 9397 20046 12547 5399 18644 11883 12531 12530 17188 2130 3805 17493 16821 10181 3639 3934 1419 780 278: 16531 9228 5799 19821 10980 17656 3449 19982 13335 20959 11238 13084 10281 17610 19623 17614 9736 21375 19978 5859 4943 12390 18806 1349 8759 21741 20400 13707 9170 15899 11361 4333 10631 12909 2136 12776 20323 7676 5847 9065 19902 4545 6768 546 9246 14134 4442 20731 9931 6700 8677 19305 4828 3655 17550 7579 21629 9044 19475 637 4209 3519 3873 15884 12247 10177 1407 10312 15957 16955 17469 20241 7267 21862 16864 22192 19599 6365 12324 10985 6424 6449 14564 3115 14885 10603 911 18609 3359 7059 2851 5801 9613 8391 18695 7987 14964 1081 15171 1312 14747 3060 13390 22115 5060 16536 19729 13468 11109 21989 2230 9462 9096 18775 10721 11999 8340 16607 22199 8687 3652 8147 22073 4090 3491 20506 7835 10890 6781 7839 14478 16371 579 6442 9721 5423 2566 7876 17580 2023 13164 9424 14096 8115 15304 14818 7282 6422 4841 10106 1075 12995 9297 21280 2309 895 12373 5366 22159 4499 737 11369 4308 13974 2794 12771 16584 9250 6930 8792 10185 17718 13148 5054 22383 9674 9868 21546 15881 20000 19029 4938 14024 15026 15535 16386 15402 21036 12653 9490 4170 11551 3711 4179 18619 16370 14161 11294 9094 2689 21881 279: 13577 10344 17371 5840 13904 12689 11795 21912 17226 14519 8854 14229 7998 8326 1400 16163 11785 10117 21511 21043 18205 2933 2043 22181 21628 20090 18018 20578 19081 11011 4768 2834 4635 13475 6306 13119 1671 21348 16926 20801 6361 4269 4366 15615 941 7046 3166 16526 6250 4790 825 16541 14270 3574 8127 18015 13147 17950 15243 6671 20662 3487 11787 6762 5488 7117 9417 15702 4859 20527 19240 17200 7115 19523 12823 9115 11770 20530 4791 20334 11019 20645 14383 1914 8945 15358 9347 2496 3098 11604 16441 19352 18451 12451 8877 15655 791 12609 12588 19785 18328 8143 22185 1488 10212 14002 16826 11779 1890 3879 20130 21633 20930 17222 2691 18852 7120 7853 644 21495 14630 21041 5676 8447 16750 10436 4523 2686 8237 2948 13559 19008 3686 18465 3895 7416 10170 18867 14574 1159 280: 16310 3796 10205 19486 13581 11022 21960 15598 12604 9662 20210 21764 8626 17323 11666 21316 6121 7438 982 2135 17499 12340 20711 6553 10296 17874 914 17716 3723 281: 20551 21096 13717 4006 19762 10017 14425 17785 6291 3855 15232 11917 21856 18564 19010 3910 13621 19087 670 10539 851 5698 9112 12694 14218 11947 18104 21227 22348 14178 1862 9829 9325 14533 13149 8679 17127 6507 10375 2782 3357 594 7548 9316 5728 7981 8022 13688 1294 13817 21893 10781 5263 11292 5492 9542 8000 16102 6328 11687 13695 15807 4068 3478 6486 13660 22165 17881 19166 3613 7013 6393 15983 17688 5124 4243 19684 17008 12366 7161 20062 12194 15870 14385 9124 9865 6211 628 6448 700 8869 17941 10697 21134 5586 4469 21167 855 20538 9251 1036 10678 1977 17337 4575 19974 3520 10195 14572 3870 21293 19011 12921 20120 11647 15054 13976 21163 20362 12988 15636 2345 4740 3205 17504 1953 15208 16834 7654 15907 8961 282: 19261 3860 7076 12616 1790 4886 9735 12611 20478 4501 18874 7032 18024 7225 4544 11443 2127 19283 7367 1338 4482 15213 20554 3826 14978 21769 11755 4250 15506 20020 6593 1286 20750 18985 16069 4571 22536 3773 11152 9745 13196 2190 8120 7914 16863 11987 16172 15399 14422 12490 8076 17180 19067 14493 13105 16459 18285 15863 14085 18130 11566 17352 20003 2995 5386 8757 19103 15685 20563 18739 20815 19454 7820 20771 7972 283: 0 284: 10750 5276 3894 3486 12240 18158 12170 15393 9765 11266 5031 2792 9334 20684 1144 13799 10858 16622 20849 22001 6897 17710 15401 18589 9550 1757 10249 21993 2001 19689 15058 4297 19990 643 11414 18208 19995 285: 17916 15231 15741 15829 4645 21977 10291 1806 21573 474 6018 2663 8036 9618 16693 3960 15864 14578 17125 15924 21826 13440 17249 8650 20159 1986 15742 19706 22092 8766 6813 17830 10853 21281 13394 5285 8139 21004 14220 17563 2086 2488 1597 4698 13233 4654 1250 15737 2907 1469 9957 13288 6516 22526 16496 14873 10471 18290 3086 11953 18592 3185 9418 17135 8081 9593 19180 4673 7979 16544 13933 1300 16782 15551 8460 15960 3405 13997 1566 21046 8636 17134 6512 6596 13346 15639 14396 9252 12093 21591 15042 6953 18637 16784 22523 6262 16933 22448 4612 19863 6076 4133 19601 3344 12192 16828 17089 19303 6118 15088 14986 21070 771 3291 2153 21234 18173 11970 21215 10644 20638 4377 21183 9519 13810 10948 17764 3781 21029 16613 18091 6526 5846 22213 22003 20765 3801 21866 21771 14860 861 6743 5007 5529 14267 14880 21391 10210 5693 5970 3793 15855 1007 13001 6878 9875 16912 19329 13614 10333 13714 6903 21112 8204 1133 21262 16852 15703 21338 6248 21547 15242 13567 16788 11020 18655 10528 19496 17440 22414 17480 8142 7760 20388 2829 16249 12914 2569 14595 7096 6689 12534 6105 16041 9242 9145 1552 10313 1379 9596 11771 5820 593 15445 3268 14744 18410 6984 10872 10053 9713 8837 1383 4305 10421 2944 20363 19120 7463 16753 20969 18430 12905 10227 11066 6057 13677 18640 4083 1527 19285 5385 17557 20851 15693 17304 1683 14391 15965 19854 8425 14916 286: 16099 8075 8242 429 13036 10217 18174 507 11062 15313 16399 14594 3820 5216 5717 10394 20464 15374 15526 18461 17556 14810 4502 12391 11462 14924 1652 18690 9504 13188 8137 3327 12056 2537 4030 1064 15390 6367 19001 8581 6859 7602 14778 7681 18172 18107 12112 7417 13896 2465 12130 19906 4949 17359 16220 13607 526 20229 16469 10705 15889 16978 8246 16554 18467 9002 9780 17882 21655 20242 21051 3977 19627 9063 10810 15051 5705 9204 11991 9915 15840 16148 14589 6171 6614 20389 6007 6226 5373 19830 9030 14898 11912 16971 18279 19602 13078 18533 1840 4491 7812 8977 13027 6371 15170 21310 6443 9755 12535 2819 14876 3052 12267 8377 3804 7983 22450 22167 22369 21674 891 13549 7113 14532 11064 9354 7858 8394 2489 17243 2044 9345 6836 2341 19148 17763 13516 14240 9032 15489 8304 7822 17083 9993 8188 5481 11010 1093 7789 21699 21306 11113 8956 18128 7880 3416 2857 17664 21589 11961 19154 11542 4812 7486 21609 5307 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10741 16309 4927 13396 21621 16662 8841 11120 14081 8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 10280 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 788 18247 9280 22095 18903 3706 15256 18593 5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 5580 18341 8588 12540 2454 3016 4970 17445 2401 11869 6193 21516 2447 10889 5190 13207 16465 9673 9771 11716 13123 18575 9584 12794 21399 20485 9218 18691 3350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376 15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 2282 14274 15956 3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 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18982 2984 11245 11137 12977 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800 7101 509 307 305 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107 14927 16006 532 428 372 307: 3349 11861 16061 5336 12949 18887 6597 3983 22099 2324 447 4512 11806 9383 17441 7957 16578 12877 12285 6745 8158 22143 14055 16201 1754 1561 18005 11717 9710 6672 1451 10276 2243 21572 5576 13128 10885 6027 17037 15496 10454 6068 15308 2899 11018 14627 20697 3974 7859 15270 15938 5811 2698 16879 8859 21272 14729 18033 840 5497 13669 21714 19513 5502 16413 15747 1844 4651 13831 22094 8195 21288 3065 16776 4822 18118 19091 5553 14834 21349 12572 16815 8955 13075 12733 4648 22049 5403 1029 1877 22486 14277 19152 1410 21143 7372 21858 17690 6467 9703 12831 4606 4506 19173 2942 1265 17141 18407 8705 18889 4746 3811 9106 17506 7669 11911 20449 10409 22212 9181 21792 22305 9003 8407 9234 9976 16656 2745 18946 4081 1944 12668 7458 13728 15650 11471 11697 10966 5324 9173 19342 21172 9379 17192 21538 8949 22434 3555 19594 13734 9786 21821 21772 21456 21196 9187 10524 11998 20944 6938 7779 8979 13537 5360 18851 21252 15297 11734 1781 17886 3931 16508 9825 5806 12904 4987 1438 8935 16959 16382 1164 22141 2007 17210 14488 6399 14781 6373 22109 599 9398 9473 5195 4504 5963 14256 6939 6345 1162 18406 17784 1442 4331 6935 5879 18326 2240 20428 19414 11272 14731 3444 1809 21833 14869 12121 5793 8845 20490 14861 12563 4528 17811 1567 9570 733 11892 13985 11612 1512 1070 2951 19948 9341 9353 1718 16198 8546 15048 9919 20674 3909 10843 16432 10218 15654 4043 14847 6714 8707 9886 20997 22088 13038 8037 9178 5352 3851 6484 16180 20444 13259 20733 21480 17651 22149 13792 13689 6933 21822 7825 13935 22010 15175 7093 21104 21571 14166 13059 1320 19388 12387 18647 10615 2572 16738 17277 10671 13485 4638 2352 7397 5048 13275 7941 11316 9127 6631 17029 14783 5081 2879 3446 12504 11878 2839 3228 19752 1088 1402 6179 16798 22525 16859 10813 2895 20050 10689 9920 13359 6382 16804 22048 15372 17024 19135 2131 20708 19186 7679 13583 7611 8668 2471 18443 8080 14254 18267 2356 17724 10645 21850 7740 18043 7128 21146 15408 8078 2828 15730 8513 18968 650 14046 3085 13243 11222 15011 1105 3976 1507 15891 10269 17937 6229 13505 13897 17262 1497 9774 5505 5939 7480 3041 12199 18054 15132 17133 9987 21835 578 22529 20727 13110 17553 15284 5186 15066 12304 21091 9863 4969 14128 13927 2253 22173 20557 7177 21588 7134 20606 17839 15095 21594 19310 4399 21404 1167 2570 8102 16155 14831 9194 9119 2558 21715 11856 11165 7573 10855 15158 22322 8996 12383 18252 20458 20237 9891 4099 16104 8881 12231 2561 647 22387 19765 4615 21305 15139 4964 20281 11812 22153 16216 18833 21797 7671 15097 11028 2172 17562 12574 2664 9816 12353 2954 21428 12735 2392 5226 8680 17177 18591 20343 15667 21341 11930 10157 19825 5210 7091 22444 3844 717 18902 14808 11659 8670 1785 8233 21693 8835 7167 12659 3869 22226 15589 13405 5280 13156 10456 10253 16881 14660 15728 6461 7132 8140 20493 14049 12322 15683 846 12989 4867 5866 20584 15020 1964 20885 21697 12785 2196 12335 15689 9927 763 1531 20138 11358 1132 19079 14162 5361 7543 18763 17112 21617 7915 20412 1643 20533 8020 9158 2930 19107 21064 3201 11077 14726 20135 9411 15348 9402 12669 15995 8168 7755 3112 12219 551 21933 8916 13876 14513 6877 8597 18116 8268 9085 9769 11890 21961 11709 10758 12800 8640 1212 10509 21023 11267 21751 1557 18735 18051 21254 15200 5771 17912 3381 18702 19564 12819 17593 8712 8128 1909 6496 14338 16091 948 17565 16614 18595 11540 18075 9306 5464 18421 10608 12167 17453 9268 11122 12880 13525 21522 5843 7885 10162 18002 13496 9474 11489 14139 5078 13260 10989 5480 3428 22424 15138 4252 2826 5882 20175 15811 16116 7879 12274 19326 5318 8325 1069 22306 6156 14294 21302 22394 4521 10893 5702 4766 568 15271 1077 17778 9726 14452 21497 5648 10532 15330 3647 8350 19227 17520 19834 16427 12852 9016 11208 9994 5906 20197 1115 15244 22452 15164 4258 6471 14545 6910 6417 21796 17471 17991 14998 15383 3215 10382 2580 8289 8452 12605 11475 6041 3868 2991 13102 15010 21365 21059 6894 21208 11757 308: 13659 8503 5417 2636 18255 14258 18186 14480 2997 12520 20393 16958 14070 11296 3421 20754 17033 17928 309: 22368 4230 16648 2237 2516 16152 10415 5473 14703 19022 18480 3997 17101 4156 10514 13955 12976 4059 310: 4051 3527 13345 2678 8550 12739 5968 11252 1249 13757 11908 13883 642 18741 863 15128 20888 14323 4672 8571 16644 10920 8004 22023 10377 10056 4837 19713 7234 1632 16113 13478 12526 19784 19698 2701 14658 15220 8365 18917 19359 21121 8529 17053 2009 2989 6906 5789 4862 8968 21524 13803 21337 5536 3377 936 20910 2445 21144 20276 15766 10716 15947 17616 7904 2437 12791 8028 5119 21686 20353 6765 13032 12566 2929 19589 6025 12404 5688 7908 7436 18460 14549 1975 9403 18910 15871 5414 21648 22373 15001 8960 8512 14839 13626 2150 18521 4687 4104 15162 19653 311: 7246 7633 13566 4922 7189 6648 3075 2678 8937 18998 20791 2442 4593 2102 13757 8484 11908 21901 9697 3719 21432 10971 8004 22023 10056 4837 6659 9021 8001 9591 4169 18319 12526 20405 19739 12836 12342 14209 15108 19698 21200 14093 4820 21147 16711 686 18917 21915 21121 8529 17053 2009 2989 6906 12853 7577 6127 14095 21524 6916 13803 7336 19598 11276 21713 14811 21337 20910 936 2445 11616 15947 17616 8331 11034 19471 5916 17862 12115 14492 18721 8414 19179 5371 9277 5119 17025 20374 3996 5057 1399 16384 9470 18719 18399 3401 8557 2158 3928 19925 15780 1172 19003 4754 6752 7557 21304 14324 15694 2929 15268 11094 19589 8713 6025 14222 14987 11178 3467 7373 1975 9403 2856 8286 16534 8056 9369 2719 15871 5414 22373 21648 1955 5457 18895 17041 22161 13812 8512 14839 13626 18521 21678 2002 4687 18560 4104 7854 18098 13647 15831 11038 19653 8205 312: 12932 12556 10612 8324 20661 21261 10886 21109 3162 13645 12519 14154 4627 9952 7890 12288 13254 22516 21541 11332 12510 6556 17293 7951 6797 12554 2010 313: 15796 4326 10345 13029 13317 20973 19570 14613 13103 3759 11764 3247 11747 9513 3029 20696 20193 9454 5495 18809 16012 8066 7668 15744 1836 6499 12779 2481 14822 314: 7479 533 507 535 306 12575 15642 18461 5073 9804 18997 8666 3659 6320 6367 19001 6157 11916 2258 21356 9857 2163 2584 3331 20086 6423 4662 3838 14933 8860 19820 15915 7403 4850 4714 9730 12137 14919 12016 19643 3913 17315 7812 21939 13951 1478 7199 10536 9491 3917 15376 4608 20333 7216 21640 8527 16880 19870 10959 4725 15069 5944 17598 6786 14232 4110 10546 16887 4811 15616 10586 10067 11756 20433 13391 16270 10468 7655 21719 5755 2857 17664 21589 11961 18800 19154 879 12644 16878 2030 4833 5428 3533 8904 4677 18511 4730 696 9569 17797 18687 7097 20259 15252 13221 450 22502 14915 21157 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292 16130 21296 2832 830 14553 6434 18295 476 12043 20779 1281 17745 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 6946 19538 14273 15340 6427 17165 15603 12419 10809 8003 13658 14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 18217 3578 22249 992 13442 3471 14855 19949 5277 19885 2816 10917 9436 18538 18659 12816 12308 15943 2515 20364 14701 8193 17579 22076 8890 2649 9329 21073 8376 15177 10882 859 5990 9814 16053 5209 3144 13848 22428 19076 14568 4694 12396 15369 16597 3697 10741 13396 21621 8841 16662 11120 14081 15204 6565 14320 2978 9294 7482 9493 1277 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 788 9673 9771 11716 18575 9584 12794 21399 6281 18662 7992 9661 19875 11156 3619 14086 786 20948 16946 3456 6143 6709 5954 17926 20839 22351 13277 10965 15558 8891 10152 15189 13161 3256 13226 1748 21271 22258 10136 18982 20726 10154 17115 12652 1426 12623 4475 16706 17850 21904 545 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 7273 14619 2800 12728 15228 19263 10088 305 16894 6203 22106 19049 10427 11733 8133 372 12866 13782 315: 3342 12667 2100 12617 1136 6158 17567 4891 9212 13652 2310 14039 8257 4890 20474 16825 8834 4127 1369 14688 17548 316: 10331 15934 2046 6293 19503 17105 13130 7604 20972 611 2151 19670 1110 20162 9143 9176 19023 19084 18099 5356 15439 3212 10361 21551 20873 18300 6915 11130 19131 17963 7910 4920 4372 18216 8334 5551 11009 21068 13009 6829 20236 2330 15785 1052 14929 4869 4064 14251 6409 4771 21722 3477 22283 4071 13419 2771 12516 2855 6554 5053 15301 13629 22091 9959 8014 18394 7184 5437 10014 15912 7764 9611 13654 12151 12808 1755 10215 21979 12492 8018 7254 20380 2845 654 5061 7274 9568 5018 11235 6804 8938 12700 17001 317: 7147 17102 22335 21107 13529 17232 12299 15442 10074 12860 8161 6451 9249 12173 13520 2381 7179 2397 4681 8032 10026 17837 1679 750 13467 22304 9093 12920 8746 12849 8975 8614 11049 15351 6414 11194 18365 22000 10060 950 10516 16860 18657 11078 20626 15349 9992 11321 16068 13071 20575 10629 4241 12058 21534 18040 574 21837 6124 1526 12392 2919 22474 21550 1309 19435 13170 2632 14407 19257 15479 17487 7797 19535 13125 7697 9338 11284 15648 2144 9885 3591 21263 9545 14344 6947 19431 21279 4756 13704 4125 11670 6044 22381 21905 15904 14499 10692 12332 1756 4806 18332 7994 9064 18991 17391 20098 22081 5015 11957 13991 17712 22029 21507 3779 6619 4824 1847 11561 2927 7497 1618 16567 1625 1011 14504 1983 2353 13906 16938 11796 2922 13819 606 3676 2458 20599 20531 13948 7520 12498 13705 8646 9042 9750 14969 563 1092 8562 6341 3562 11565 15599 16895 12974 1939 16577 1711 21985 5754 19800 15748 4371 12415 2463 4827 5656 6520 14881 1513 19711 6790 20528 13821 9526 8611 6058 17821 1888 18587 4858 18855 7028 14908 22205 5964 4340 5271 17773 927 8374 3316 2666 20014 21527 9914 10822 7197 21212 13163 13989 17347 15266 1401 6338 14444 3445 561 9349 20565 2095 7453 18711 19524 4093 8735 14800 9217 16762 10080 19195 11057 11977 21372 20196 6610 14503 22133 19181 318: 4562 9606 1444 14309 10107 21463 18996 14333 1334 13632 22339 19891 15309 3134 19973 17693 15433 13252 20233 7289 15373 8208 21182 21283 14523 6862 8920 22389 7427 19075 14622 18190 16360 12132 1586 14883 3538 22299 4909 6348 9295 11703 6913 16728 19809 21668 18897 20235 616 10625 12272 10812 4295 2449 11240 20715 10788 12331 319: 11506 8025 19504 17096 9236 5967 13265 13150 20345 2575 15093 720 9238 20846 7110 18462 16689 3666 13238 20249 22504 5681 15821 11308 7263 2587 3204 8488 1135 3170 19880 7687 4736 5783 20141 8454 11622 3641 10121 15474 15016 3635 12476 2148 6474 12754 10278 4251 2157 17396 18150 5472 2435 9967 3440 12802 21724 16131 7711 10389 5452 18194 15428 5298 16650 13697 3148 15338 9355 4542 7607 12624 9835 6871 1450 17202 4346 18156 2256 5469 11562 11593 8125 13465 21018 14122 6978 1534 5284 19934 13992 9438 9860 16616 13159 22237 9239 12122 7442 20605 16637 625 10834 2546 3875 5692 13941 320: 5584 14500 1413 1303 11425 12432 20553 5678 19728 1702 21840 16132 13978 8053 11638 17005 7048 12602 12897 18092 1532 16921 11347 8929 2022 22014 21806 20100 17247 5940 13718 10507 20917 13044 3334 11629 3850 19276 8082 15982 2091 8247 2827 4647 21352 16763 13875 2729 11068 14988 22366 6081 6500 17279 7348 9062 12873 13199 7794 6832 16726 7795 12202 12467 5788 10596 12756 21948 11263 10951 13316 8432 13127 9984 13493 8019 12038 1826 14472 9125 4955 19548 16684 5905 16785 3169 20313 12232 10211 6536 20084 4322 19294 321: 11679 7712 4765 2778 10428 15613 13759 8748 15529 4789 4143 22492 10520 5899 5524 1150 15997 6728 7250 19339 3882 2486 16390 13804 4361 20834 20825 586 2032 2861 14363 17505 15033 6284 9806 19295 8378 20979 6962 22356 6126 18081 16902 16016 11797 14905 12294 1932 4974 560 4872 19916 7886 18272 11004 21567 6122 3929 10292 20964 14636 4316 16294 16670 6202 3283 3979 708 10683 3722 9024 10315 7707 21597 12187 21031 11517 17433 1323 3512 20375 12464 21941 10366 13212 15089 3993 3918 17676 18119 2709 17107 15788 322: 10548 4897 5425 7818 3878 10642 3310 16365 9020 12789 17219 2142 13865 19002 946 18211 323: 12932 12556 10612 8471 9836 7200 3244 4931 6155 11441 5925 9952 1283 20609 2129 3224 8050 2693 11630 8765 8258 10271 17801 10768 7008 3010 16997 6501 20840 3978 2010 324: 10963 8235 5742 21966 17140 325: 15756 6758 15758 5287 19343 4588 17303 13336 21407 16436 6718 326: 16712 1166 15865 2733 17246 15303 1381 13492 6572 7728 13605 12382 9290 21152 7868 21359 6654 2544 327: 12913 16464 16273 20905 3701 8623 4539 11237 7785 12606 12734 1726 4555 7538 13748 14436 18787 11199 15234 14408 15929 7578 1919 8057 11210 328: 14125 20217 16758 5776 15086 18339 9344 13845 5197 20795 5897 9137 9973 704 18058 11958 7252 2114 7827 5752 4689 7592 15102 22013 12505 15413 5397 16345 11067 11445 21494 14366 3450 8189 13758 10214 21577 10815 1385 19042 329: 3497 2714 8101 4216 8074 1106 1600 330: 11223 1913 6535 17340 17586 15949 10628 5279 16989 13504 22441 20437 16847 20368 18727 20627 677 8442 2847 12004 5478 331: 13633 5900 8539 14672 12233 964 10478 20775 5875 5805 1804 1598 8760 4906 10137 12276 20894 3902 1838 1416 13999 8927 20239 21067 2846 1536 951 9346 9547 21436 4596 12781 4010 11786 8601 10761 6187 19637 20291 12933 1404 16680 16262 4172 6985 11107 10370 17700 13722 7971 3488 17731 14386 6750 13862 2765 17564 11582 6358 16691 21558 16323 14350 16234 10634 8984 7275 21757 12586 15643 921 10884 20529 8362 4086 15778 8213 645 8641 14759 21903 9293 2872 7350 12282 7277 21911 19806 10064 12113 15339 20238 1645 15286 17911 15808 9799 12685 4914 19213 10728 2852 4839 20536 7995 7379 15240 12971 591 20690 5666 5145 10145 1126 13494 13052 13798 22187 6567 2224 22255 22276 19919 8963 4880 11857 15344 20168 20601 21712 7009 15363 10724 8126 7044 2098 12067 13898 7690 17967 22554 332: 4051 7246 14199 4757 2211 19939 7119 18627 18219 10672 19725 20509 7686 2989 10561 7284 936 20910 3464 21563 5884 11537 21063 1972 3103 11411 17085 21614 20041 6920 896 19271 21101 6679 18617 19003 7557 2937 8512 333: 11864 18401 7916 16388 781 8033 2364 22015 9748 2475 765 14146 13993 16445 7070 19406 13139 11260 21048 22437 16423 12388 15277 9405 13452 21701 15472 21519 18268 18140 17189 10852 15461 8741 9641 20297 21384 9522 16319 4702 6436 9574 18726 3628 16593 18634 13820 16639 4189 17966 3524 13210 12214 13114 18483 11648 5974 15580 4570 17225 19309 11814 7466 15434 16431 13455 9128 17770 17597 11744 5668 18077 20332 12252 6487 5722 19526 13902 14620 19013 12349 14056 3612 8715 15923 4913 11247 1777 16114 9983 8124 7153 20006 10025 1074 21879 4977 19631 8517 5456 9877 4139 21162 4364 7696 9702 1769 9365 5341 7159 5323 19419 19967 19719 15541 14191 10391 612 17071 22520 2596 16797 826 17040 18360 6069 16301 22129 12523 19596 3074 6712 12666 21205 18284 21965 334: 22427 862 20385 6265 12399 22070 17740 7828 4067 1153 11326 2935 9841 19586 16621 4285 4474 5816 1468 12636 2870 12256 15396 20978 13936 20481 9196 15850 17759 14083 17877 11918 14099 22052 4350 14967 3030 4936 6573 20255 16179 17938 6661 14748 18841 8544 17559 16186 13096 10817 20956 15131 16981 20307 6896 9262 16287 14784 4658 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14077 9240 11996 3702 14090 19402 7233 5546 10148 20680 8638 3269 15282 9663 15009 15604 8648 14392 18782 10570 6318 19862 13514 13217 3980 5231 11578 4812 18337 3004 3073 8249 18941 9036 3233 864 15080 19930 6746 1476 10866 20483 19942 7450 762 21276 3235 15320 22462 11955 1562 13099 7710 9136 7251 16129 19467 10892 10630 13600 12857 14732 4581 18096 3518 12368 1009 17283 4039 6864 5773 6063 15842 16851 18149 7977 14608 4572 10553 4514 11848 18068 17157 12223 18597 15178 10550 11835 16242 11778 20884 16907 14445 5068 7404 1516 6277 17663 17662 21513 5526 14789 7565 21347 1238 10660 18496 16381 20827 19639 528: 22368 2262 4500 18087 11171 3257 5531 17538 11849 9017 9287 9644 19677 20195 3911 3945 21129 6446 529: 18280 2326 4826 11407 11186 16051 1917 13113 10636 10206 19238 18366 6941 11986 7683 16587 14340 18850 6892 3404 17954 20223 11450 10435 19074 13384 7518 15985 8336 5383 3907 13379 12855 14509 12217 3398 530: 1719 15257 12828 16494 8849 18495 10061 1831 11005 20446 21892 17075 9052 1023 10386 2348 6364 14482 15814 7746 9263 3529 9935 10786 20919 12407 11705 8493 5095 8481 6104 3104 14770 19267 1032 17399 8085 17728 7344 673 15714 3361 5103 21218 15350 7539 761 19758 16925 17515 8371 9594 13769 16744 17772 4420 8605 6819 6628 9541 19100 13982 7360 20665 9903 5639 8487 13488 9666 5511 11106 17709 5021 19088 20007 12811 14758 15329 7823 21543 20508 1178 5558 16935 1252 7312 20192 18963 5305 19575 17193 1262 3577 20951 2711 18829 9159 6009 6176 17751 9694 17992 12429 15448 4598 18452 7077 10597 16197 705 8820 11971 19192 1370 996 1578 21971 6312 17349 2736 3936 10254 10398 22265 20083 15853 14066 5564 9113 3087 1078 7435 14028 16372 9321 18704 8983 21564 9660 11558 17282 12695 19057 13847 17094 11400 7613 2232 17213 17103 19378 6072 14868 8879 6825 12193 1427 3275 9458 15007 6271 4411 4190 12430 16366 20438 15680 10929 4035 20030 4556 17229 4660 597 1587 15707 4334 22484 21035 8211 16251 13133 11295 2966 14058 5665 6592 8738 6102 13087 3436 12074 14546 13772 20692 10970 19345 13509 5329 19187 16282 9968 21671 9048 8976 7492 21462 19662 8457 8317 18846 12864 10746 4141 8673 21010 10417 4329 7895 998 10132 8852 14795 5620 6695 13781 19165 7258 20895 4154 7359 12259 11737 12704 21606 3865 5520 6932 12901 20940 15027 3236 4907 20734 15492 10607 2472 15046 8506 18724 11671 15259 15936 7889 5596 2053 6905 8323 16351 10263 15826 21160 19461 17456 3953 3584 4314 6428 14221 4758 5800 22365 17678 16252 14893 17154 19437 16453 21224 9572 16225 9367 1899 8751 8426 12483 4441 20592 6867 2168 16928 13225 22460 20208 12970 2591 5304 13498 21581 4699 21894 1461 5860 21622 18703 2500 7014 15116 596 14962 5517 13693 21739 19681 11533 3338 3956 11364 17988 531: 15910 22524 13589 1006 11575 20349 18523 10911 13067 10217 9609 5233 4966 9015 21390 18461 9866 21665 7323 5049 9997 5240 6367 19001 2814 21443 1880 1396 3615 13867 7787 22538 13411 13344 1205 18398 13083 17485 1550 757 11281 11029 21967 827 1873 21174 14091 5281 885 7332 8229 21103 4400 1012 15883 7812 7121 16424 1339 1866 3474 11617 19918 14296 1352 17699 12367 3388 20896 20167 6697 19989 9358 15375 4618 11468 13543 3097 15540 19380 14287 5051 11086 12824 13271 15196 6303 4194 1182 15803 1842 19299 2857 17664 21589 11961 19154 12506 21814 6334 21437 20936 11195 20933 21506 22069 5476 3533 19583 7949 4677 18511 4730 696 9569 17797 18687 7097 20259 15252 13221 13305 450 22502 14915 21157 6730 2195 19855 10324 11089 1744 19917 16130 21296 2832 830 14553 6434 18295 17956 829 476 12043 2457 13959 12384 5220 3209 19341 5731 10908 4395 21843 14568 17630 2371 4694 12396 15369 13051 16597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081 19449 21259 15204 6565 18317 14304 4952 7813 7306 12296 12021 6645 21386 16505 9286 22489 3622 14762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 3836 7637 4946 18143 11559 18362 12059 10889 5190 13207 16465 17346 3323 5354 5825 2210 19147 9333 13177 20541 3458 1240 15787 20099 9282 11480 4994 14011 1145 19545 802 17144 13420 13439 14301 1357 10965 15558 22251 22565 810 7570 2692 15666 13168 10912 20574 4894 2526 18982 4020 19796 2984 11522 11245 21570 8695 16749 17392 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800 16658 18348 16408 4157 8112 5264 305 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 3397 11197 13107 675 16006 20604 18536 532: 15542 3190 1006 429 7236 21108 10856 10217 1694 1170 507 478 7946 4963 10952 8465 18461 9739 1850 5240 2382 6367 19001 19407 14319 21808 15520 1498 14212 14282 19450 526 3987 18034 12939 14517 22533 14740 7812 18725 15422 12515 6665 8294 20640 9749 7933 18742 10601 1528 14184 8297 11430 21253 7897 4901 7308 16574 19527 9882 8330 6186 16730 3076 15579 16073 19400 17335 2857 17664 21589 11961 19154 6997 3197 21437 4440 11195 20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097 20259 2920 15252 13221 3452 14930 450 11335 20777 12713 22502 14915 21157 14054 10864 5059 21184 8816 9146 14653 14579 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292 7706 5100 4540 16130 21296 2832 830 14553 6434 18295 17956 829 476 12043 13241 11023 4301 9298 2269 1306 13291 17064 20779 5613 1281 17745 11486 9723 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 20461 6946 19538 14273 15340 6427 17165 15603 12419 10809 8003 13658 14064 14568 17630 2371 4694 12396 15369 16597 3697 10741 12482 16309 4927 13396 21621 8841 16662 11120 14081 15751 4903 21508 18755 14599 6384 8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 1733 20453 21787 16624 7001 20828 13302 10357 4549 788 10490 19133 12318 5515 9280 22095 18903 3706 15256 18593 5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 5580 3044 18341 8588 5857 12540 2454 4970 16919 17445 2401 11869 6193 21516 10889 5190 13207 16465 9673 11625 9771 2283 11716 19554 905 7481 12401 19686 13656 22135 9553 18575 9584 12794 5644 21399 20485 9218 18691 3350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376 15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 2282 15956 3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 13940 18821 14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 13014 14274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 21928 13800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 1429 14398 22189 6439 12993 12307 19714 6650 18994 747 5932 18630 6683 1921 15651 5594 6958 13597 19763 10097 14124 14687 1094 5780 7770 7688 15110 5797 7907 21169 3329 12627 4065 19882 22067 19211 2061 7038 8909 16914 13129 19881 11720 16317 1551 13235 14888 1201 20264 7800 7526 2209 5887 15285 10798 15317 10223 11351 10105 10661 15014 2908 16412 22277 19851 12003 19616 11003 7768 6166 21613 4620 13850 16231 7016 2717 20541 3458 1240 15787 20099 9282 11480 4994 937 16775 18459 14476 9221 17450 6281 18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839 22351 13277 11255 5140 4283 9455 4818 14459 5187 16668 10965 15558 17215 14289 11685 4388 14119 18982 2984 11245 304 477 427 18112 965 2900 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 5498 2800 19337 9600 307 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107 16006 7623 533: 6090 1006 429 10217 10250 19910 18933 314 18461 5240 6367 19001 16913 19119 526 3693 7812 13179 534 300 531 2857 17664 21589 11961 19154 21437 11195 20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097 20259 15252 13221 450 11335 20777 12713 22502 14915 21157 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292 16130 21296 2832 830 14553 6434 18295 17956 829 476 12043 20779 1281 17745 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 6946 19538 14273 15603 15340 6427 17165 12419 10809 8003 13658 14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 18217 3578 22249 992 13442 3471 14855 19949 5277 19885 2816 10917 9436 18538 18659 12816 12308 15943 2515 20364 14701 8193 17579 22076 8890 2649 9329 21073 8376 15177 10882 859 5990 9814 16053 5209 3144 13848 22428 19076 5454 17056 14918 4088 11270 5837 9604 22455 19840 12234 1149 801 953 9960 21301 1656 3755 12278 22119 21098 1005 10264 13037 11702 20954 5581 5063 1118 6454 20632 1696 19474 14911 9381 4111 13374 2932 3071 18551 22362 1431 12923 6509 19229 14012 5372 12362 17380 20272 16391 13395 5132 901 9540 19228 11589 13306 3179 14568 17630 2371 4694 12396 15369 11784 16597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081 8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 10280 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 788 9280 22095 18903 3706 15256 18593 5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 5580 18341 8588 12540 2454 4970 17445 2401 11869 6193 21516 2447 10889 5190 13207 16465 9673 9771 11716 18575 9584 12794 21399 20485 9218 18691 3350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376 15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 2282 15956 3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 13940 18821 14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 13014 14274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 21928 13800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 1429 14398 22189 747 6439 12993 12307 19714 6650 18994 5932 18630 6683 1921 15651 5594 6958 13597 19763 10097 4065 14124 14687 1094 5780 7770 7688 15110 5797 7907 21169 3329 12627 19882 22067 19211 2061 7038 8909 16914 13129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209 5887 10223 10798 15317 11351 10105 10661 15014 2908 16412 22277 19851 12003 19616 11003 7768 6166 4620 16231 13850 7016 2717 20886 14522 5254 15519 20541 3458 1240 15787 20099 9282 11480 4994 6281 18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839 22351 13277 15113 6368 7704 18694 3644 12154 3794 21257 21638 18386 18111 21498 10731 13776 5539 14530 20282 7762 9529 17675 15191 12380 14865 15825 2818 16442 5901 8220 18578 13297 2495 21913 4526 16085 10965 15558 18982 2984 11245 21040 477 11140 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800 15563 509 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107 13802 16006 428 534: 6383 10450 1006 8266 17986 13269 9010 9822 18387 3735 10217 533 535 1774 16191 18891 13849 14706 571 8356 3340 18461 20284 20753 10192 13836 5240 2653 20833 3564 1870 11250 6367 19001 20137 15672 13176 20207 8314 4015 1887 8795 21466 7023 18171 6876 8560 16477 13630 10793 4136 5249 17099 9152 2730 4724 8684 5821 3710 16562 22562 9500 21952 12129 1151 5863 2635 13579 5947 9543 6219 4851 7812 10216 19130 12258 1950 8843 1084 2519 12495 7141 1845 6128 9067 2618 1308 1202 16045 2887 3970 22176 2992 14189 2020 3687 18669 17082 7279 10946 10754 10583 4481 915 16903 18701 14442 11182 12710 8108 4737 8174 21990 14565 20445 20022 9861 2888 17080 6216 3441 9629 4034 4019 17401 1207 22219 1481 5143 2857 17664 21589 11961 19154 12425 21437 17489 11195 20933 21506 3533 12843 4677 18511 4730 696 9569 17797 18687 7097 20259 20966 15252 13221 11335 20777 12713 22502 14915 21157 13596 7735 6111 14062 20371 3563 12591 16308 7626 21483 4212 19798 19458 5691 18292 16130 21296 2832 830 14553 4632 6434 18295 17956 829 12043 1189 3932 18731 6845 21360 6772 19039 21673 20779 1281 16128 14568 17630 2371 4694 12396 15369 14260 13396 21621 8841 16662 11120 14081 6657 8692 9484 14065 2540 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 19908 893 14762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 2081 12643 15099 7425 15838 10094 788 3348 9280 22095 18903 3706 15256 18593 5764 11115 12583 5630 11568 13613 2331 5136 8073 15998 11304 19137 5817 4430 5580 8588 18341 12540 2454 4970 17445 2401 10889 5190 13207 16465 11291 16479 21467 4263 2705 9673 20541 3458 1240 15787 20099 9282 11480 4994 2798 5475 1814 2683 8435 4345 3627 15897 9366 6281 20716 18662 7992 9661 19875 11156 21818 3619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839 13891 10965 15558 15276 3513 17600 11426 1123 16786 5326 17407 18982 2984 19817 11245 5838 21039 15310 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800 16353 4982 22032 3731 7388 13436 8698 509 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 15886 11197 13107 5444 16006 9573 20805 372 19913 9499 18057 7973 22459 21704 8464 535: 9382 1006 429 10217 2438 9883 314 18461 5240 6367 19001 16674 12484 526 11896 7812 9819 2857 17664 21589 11961 19154 21437 11195 20933 21506 3533 4677 18511 4730 696 9569 17797 18687 7097 20259 15252 13221 450 11335 20777 12713 22502 14915 21157 20371 3563 12591 16308 7626 4212 19798 19458 5691 18292 16130 21296 2832 830 14553 6434 18295 17956 829 476 12043 20779 1281 17745 16586 7955 12705 15449 988 20859 1768 11129 6547 22518 5552 9854 11376 6669 5359 9132 10964 1879 22087 13137 17298 3333 13752 20301 19370 2425 6946 19538 14273 15340 6427 17165 15603 12419 10809 8003 13658 14064 4221 7387 13284 20981 2316 3116 8418 3386 6508 18217 3578 22249 992 14855 13442 3471 19949 5277 19885 2816 10917 9436 18538 18659 12816 12308 15943 2515 20364 14701 8193 17579 22076 8890 2649 9329 21073 8376 15177 10882 859 5990 9814 16053 5209 3144 13848 22428 19076 5454 17056 14918 4088 11270 5837 9604 22455 19840 12234 1149 801 953 9960 21301 1656 3755 12278 22119 21098 1005 10264 13037 11702 20954 5581 5063 1118 6454 20632 1696 19474 14911 9381 4111 13374 2932 3071 18551 22362 1431 12923 6509 19229 14012 5372 12362 17380 20272 16391 13395 5132 901 9540 19228 14568 17630 2371 4694 12396 15369 16597 3697 10741 16309 4927 13396 21621 8841 16662 11120 14081 8692 9484 15204 6565 14320 2978 9294 7482 9493 1277 4952 7813 7306 12296 12021 6645 9286 22489 13745 16653 19069 7780 15219 16969 14762 18330 10802 10479 16663 3678 16713 7751 13703 3630 4691 9472 10709 8542 7060 6112 22457 21974 20476 7333 6482 14526 7151 2644 835 10655 12264 9315 2786 16253 9488 5634 17372 788 9280 22095 18903 3706 15256 18593 5764 11115 12583 11568 13613 2331 5136 8073 15998 5630 11304 19137 5817 5580 8588 18341 12540 2454 4970 17445 2401 11869 6193 21516 10889 5190 13207 16465 9673 9771 11716 18575 9584 12794 21399 20485 9218 18691 3350 18263 4846 544 19667 9933 4140 1318 20418 11128 20105 16734 2376 15699 7061 4232 15357 14036 5339 7107 19030 7165 21370 12103 4848 13211 22530 15360 12863 9975 6398 14067 16683 21170 1924 890 8155 15866 10131 7187 4332 1235 20330 12927 7088 5099 2302 12424 8303 17466 14322 11383 2282 15956 3414 12982 18548 15665 10961 21084 10824 18440 16819 3730 13940 18821 14864 1818 19607 7969 6546 16771 5441 8459 18266 5000 15749 13014 14274 8444 4707 13097 15930 11872 2621 7158 6942 18502 5408 10837 21928 13800 5188 19614 16117 4719 1833 13499 3107 21492 12503 21929 7921 1429 14398 22189 6439 12993 12307 19714 6650 18994 747 5932 18630 6683 1921 15651 5594 6958 13597 19763 10097 14124 14687 1094 5780 7770 7688 15110 5797 7907 21169 3329 19882 12627 4065 22067 19211 2061 7038 8909 16914 13129 19881 16317 1551 13235 14888 1201 20264 7800 7526 2209 5887 10798 15317 10223 11351 10105 10661 15014 2908 16412 22277 19851 12003 19616 11003 7768 6166 4620 13850 16231 7016 2717 20541 3458 1240 15787 20099 9282 11480 4994 6281 18662 7992 9661 19875 11156 3619 14086 20948 16946 3456 6143 786 6709 5954 17926 20839 22351 13277 15113 6368 7704 18694 3644 12154 3794 21257 21638 18386 18111 21498 10731 13776 5539 14530 20282 7762 9529 17675 15191 12380 14865 15825 2818 16442 5901 8220 18578 13297 2495 21913 4526 16085 10965 15558 18982 2984 11245 16474 304 477 427 5067 2132 20312 4655 16568 21093 13882 5730 7976 7079 13035 2800 20416 509 307 305 12545 2037 11704 9571 21379 3712 7685 4483 18456 22341 11197 13107 13268 16006 428 536: 8250 5293 17519 10969 8775 16532 10286 11811 5689 2871 680 20415 14104 16888 17043 10273 6777 22396 602 4866 12333 9909 22327 4344 9005 16476 2573 10616 19638 15657 8950 4378 2657 3364 21708 985 6316 8776 2238 13856 1099 10466 19656 6879 9313 19604 12069 8122 21385 16087 20487 1424 14078 2427 4175 7418 9385 16892 15809 3499 16714 5768 6322 13627 19469 8252 19551 14976 12742 10499 11322 9597 3569 9336 20132 5283 22127 10381 3149 7067 13886 7478 3897 20869 19398 13086 4191 3669 20800 6799 8453 17539 21956 5724 13878 16687 1797 537: 12225 7991 19769 20707 6346 21228 18920 15677 22007 17216 1246 1299 3482 8428 4864 1869 18259 5076 18894 10621 1194 21241 4280 10372 9939 3461 22101 14388 9687 1934 3266 19426 6888 17781 1697 1251 15687 11467 11444 7248 4960 3598 18026 17460 7270 18737 20522 18581 16569 13403 4999 20923 3253 12847 6541 12065 13811 442 18246 10039 21176 20831 13532 19077 10287 9635 12724 898 4347 11482 12691 8702 12055 8006 14571 19675 16542 21229 14891 11459 15395 15872 9301 10051 6843 5509 11707 10865 18908 12031 9844 20469 4041 4623 22346 1849 12095 14281 3840 16733 15322 21786 17305 10611 1428 7631 22045 6676 5894 8015 21902 14896 17330 3090 15122 7759 6196 16678 475 20224 3032 21119 21396 1165 20759 22152 18157 1337 3062 12222 10847 14596 3679 21087 17626 3743 10092 21220 22333 4831 6340 2684 14394 17353 12317 2073 22147 10166 9664 11412 21185 7389 11862 11526 9220 2983 5429 13258 10529 12270 6868 5672 2355 11711 9314 17713 6624 12386 17898 3480 10028 20275 1292 12254 17594 20642 4853 10383 4591 8186 16379 15333 9201 15765 17720 5294 9934 7376 4038 2297 1916 7144 16736 10304 3319 3762 20614 11341 10440 4734 9677 12289 20772 4271 19954 22175 3020 19748 16083 8948 19737 22114 2955 21137 3274 1408 6780 10848 7124 14060 11449 17939 4794 16058 21761 10251 4961 19141 1661 9701 6223 5983 18048 10670 17196 1709 4904 12196 630 8403 8582 6184 18819 3380 9202 16022 17350 12703 15237 17792 4597 11159 1072 20441 15476 15612 17341 12261 21044 4892 1108 1406 22046 18198 6967 20318 12469 22123 7645 10452 1264 712 14602 6109 14712 22157 7745 16695 11050 22271 21574 538: 8633 21066 20486 15659 19893 20341 18625 7202 9210 15561 8243 19452 10486 9599 20104 5911 17836 3744 3313 22371 13393 8083 14598 8878 4121 11696 16024 6949 9760 18073 21021 1691 3955 13639 14076 5677 2739 10860 20215 14300 10639 13585 11393 3392 7331 2940 4631 3646 14037 5709 8396 7169 3234 9515 13640 21141 9848 13213 16854 10018 17683 4171 20607 15041 2557 19483 17558 19443 20760 13774 16065 5097 13747 713 8505 18788 18352 817 5302 8167 10335 22171 13497 18845 11474 8093 22332 17185 18392 15387 7019 17613 13932 22281 9232 21810 19046 14214 6221 10362 21188 12117 2909 9179 16519 21842 14950 5628 5467 18729 7240 8434 11000 2806 17205 1863 9645 18886 6782 20838 7708 6070 7788 22286 19842 16891 9527 18008 7791 11889 5849 2006 3823 12758 17857 15410 11552 11047 5550 18384 903 18870 12346 18758 1298 11723 11231 13187 11334 10943 11898 11754 4492 6688 19935 13184 13651 1487 20841 6884 6464 8803 16050 6362 1430 1363 10992 6297 3031 2620 18570 22021 21438 13674 22415 1440 6599 17348 16403 2064 5725 621 12956 13371 3689 813 8410 17547 5880 7069 7272 13965 9296 18273 16456 7801 2112 17834 8758 7378 19838 6388 10579 19671 16279 806 14835 2530 4277 795 12076 5622 13454 3935 4559 18529 18155 19209 15646 5247 6539 16369 5499 21300 14158 12126 17163 16184 993 21342 16623 17172 10687 11363 1456 21186 21282 21603 10623 6475 17459 18745 21602 9996 3042 16551 1505 5224 5300 3833 4119 10925 11838 21509 15409 13868 11753 16835 13881 7206 18880 6510 4201 3942 968 5155 13073 10765 5458 12673 3180 3108 15770 908 21482 5413 18381 14279 4351 15582 22158 16937 12412 13635 14711 5969 9888 21962 11801 16194 10513 4703 11781 15593 17383 18258 9184 7699 9109 1971 18780 3171 11419 10405 631 15583 21710 7483 5744 22254 12810 13894 20089 6909 5948 3120 15669 17412 19244 2016 16850 22002 7260 14202 3293 4461 2271 14582 18598 17872 1569 18470 5671 8965 4082 7327 22511 15167 1160 15734 5450 19432 14387 14587 3176 8359 18160 11357 15085 6948 5337 2567 10321 22182 10881 10168 18477 12537 19015 20260 2170 18422 2440 3225 5484 1260 1822 7532 19248 7693 4353 10441 2660 5734 10038 19976 3453 19019 1674 12312 16581 10232 3727 1523 2140 22432 20580 9947 19324 20180 1384 2325 18901 10198 20110 9744 9208 3182 14144 18875 14329 929 22054 2752 4524 986 977 21117 9567 9679 12352 20957 4772 12912 19652 11510 5608 21827 13765 8183 14331 13670 20503 12206 21116 22320 844 2226 21795 13733 11051 20995 2615 7715 4478 2290 12549 15082 2491 2301 14182 15076 1227 10071 3792 13186 4722 1595 10880 11269 8528 3213 14537 4257 8761 17108 4834 2651 19705 3355 7552 6681

Example 3 Consensus Sequence Build

ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 379 and 10 homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. Attached are the sequences of SEQ ID NO: 379, its homologs and the consensus sequence SEQ ID NO: 22569 at the end. The symbols for consensus sequence are (1) uppercase letters for 100% identity in all positions of multiple sequence alignment output; (2) lowercase letters for >=70% identity; symbol; (3) “X” indicated <70% identity; (4) dashes “-” meaning that gaps were in >=70% sequences.

SEQ ID NO  2406      --------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQQQQE- 15414      --------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQHPQE-   587      ------------------------------------MMGRVMEKPSQDLLQQQQQ-----  9696      ------------------------------------MMGRVMEKPSQDLLQQQQQ-----  5895      ------------------------------------MMGRVMEKPSQDLLQQQQQ----- 17251      MFGNGNCDVDNEKTIITSSKWTQSEIDDHKVSMASSTGNRVMEKPGQELLQQQQQ----- 19549      -----------------------------------------MEKQGQELLQQHHQQQQQQ   379      -----------------------------------MQSKNMIVASSHQQQQQQQPQQPQP 21357      --------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE 17711      --------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE 13715      -----------------------------------LTLTKCCMQRGSHFRSRSGSQEARS consensus  --------------------------xxxxxxxxxxxxxxxxxxxxqxxxqqqqxxxxxx 22569 APKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRRNKRVKRP APKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRRNKRVERP ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP ALRCPRCDSSNTKFCYYNNYSLTQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRLKRP ALKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGYRRNNKRSTS QLKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGSYRKNKRVKRP SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVKKS SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVRKS GSSMSRCNSMDTKFCYYNNYNVNQPRHECKNCQRYWTAGGSMRNVPVGAGRRKNKHTGSV xlkcpRCxSsnTKFCYYNNYslsQPRHFCkaCkRyWTrGGtlRNVPVGggxRkNkrvxxx LITTNPSSAAIDTAASNNSSN-SSSAPLQPPIDTASTS--------------NHINPLFY ITSPCSAAIDTASNSSNSSSAPTAAASLQPQIDTASTS--------------NHINPLFY TNHGDSSSSAANSPSSSNSNPPSQPHLDNIIASSSTTN------------HINNISPFFY TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY TYPCSNNNNIDFSASPSSSTPSSVVANPNPPSQSQQQQQQQQHHSFDIAATSNHINTMLY SSNGPTSTTTLIKRPISTIETATTSNSSSPSSTHSSTS--------------NHMNPMFY ------STATTTTASTVSTTNSSSPNNPHQISHFSSMN----------------HHPLFY ITPITTSSTTTTPITTATSTCTATVTTSIGNNNNNMDAMLG----------CYSHMTIQT ITTPITTSSTTTHQSQPPLQLALPQSQPQLATTTTTWMLCW----------VVIAT---- YRHTVITPDSLASLQVDGPDLVDHKPLSPFKVNGTILKFG--------------PDAPLC xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx----------xxxxxxxxxy GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGFSSGVLSNEASDNNGYR- GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGFSSRVLSNEASDNNRYR- GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN-- GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN-- GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN-- GGNSCH-DVMNFPFSTRFNSTTRVSNPASGYDNLPQNGLGLGFSSGILMSAAGGEVNLNH GLSSTNNPCDPNLPFSRFNITSRLSTSSGYDLQPQMNFFGLGFSSGFENNGYTNGFNTS- GLSDHMSSCNNNLPMIPSRFSDSSK-----------TCSSSGLESEFLSSGESSLSALG- PLADDQKNMSSSLYQALIRPPPLLLQQQNLLNTRELEGKDFGIGIGNGNNGIFPSSTLAL ------------------------------------------------------------ ESMASILNLGEQNLSSQLDFTAGAE-----------NREETSCSSACKPVKKKDITQHN- gxxxxxxxxxxxxxxxxxxxxxxxx----xxxxxxxxxxxxgxsxxxxxxxxxxxxxxx- -------------NWFGSNNTLLSSYTSTTSTTTPAMSSLLSSSLLQQKFMTDGVD---- -------------SGFGSNNMLLSSYTSTT-TTTPAMSSLLLQQKFISGGLKNDAD---- ------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQNLMGGGG---GG ------------SFESAYNSMEGSSSSSTCAPSTPVMASLLSCTLLQQNFMGGGG---GG ------------SFFSAYNSMEGSSSSSTCAPSTPVMASLLSSTLLQQKLMSGGG---GG HHHHHHDEGSYRNGFSTSNNNNYSSIFGSSSTTTPVMASLLSSTLLQQKFMGTGGGIKGG ----------------NNNYDSIFSSSTSASNNTSVMPSVLSSTLLQHKFFDDGLK---- --------LGLPHQMSHDHTINGSFINNSTTNKPFLLSGLFGSSMSSSSTLLQHP----- P-------------IPHQSQSLLFPFSASSRSFDTNPCSVVSTSLRSSNVYNYGED---- ------------------------------------------------------------ ------------------------------------------------------------ ------------xxxxxxxxxxxxxxxxxxxxxxxxxxsxxxxxxxxxxxxxxxxx--xx --------STNTFQHGLGLTPLEQLQMASDHSSEAGMVALKDVKVELGQNNRLEWNNGAA --------SSNTFQHGLSLTSLEQLQIASDHSSEAGMVALKDVKVELGQNNNRLEWNGGA VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG EGEEVVIMIKVATLSMAWHRYKGCKWKGIIIIVIILAQKK-------------------- GGGGGGDDDPFHHHQEMDSKEVKLGEGLQNRLDQWNMNNLNGNGGAVFQNQMENMGLSDN ----------YGSDAGSNGAFQDLQFGSKMQNQMEHIGGFYDPASSIYLNATSSSAIGVW ----------HKPMNNGGDMLGQSHLQTLASLQDLHVGGNNEDMKYKEGKLDQISGNING ---------QFKAIEEPTINSTTATIVPSTGGTNNTHHPWEIAAATSGVGLGTSSNSNYW ------------------------------------------------------------ ------------------------------------------------------------ xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx CQSQIQHVGLYDPSLYWNNSAATALGVWNDQAANIGSSVTSLI FQSQIQHVGLYDPLLYWNN-SATALGVWNDQAANIGSSVTSLI GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPNNIGPSVTSLI GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPN-IGPSVTSLI ------------------------------------------- NASLYWNNNHNNSNNNTSA-TATGLSSVWSTDQPGSNSVSSLI NDQGANNIGSSVTSLI--------------------------- FMSSSSSLDPSNYNNMWNNASVVNGAWLDPTNNNVGSSLTSLI NWEDFDSLVSTDLKDPWDDSDIKP------------------- ------------------------------------------- ------------------------------------------- XXXXXXXXXXXXXXXXXXX-XXXXXXXXXXXXXXXXXXXXXXX

Example 4 Corn Transformation Construct

GATEWAY™ destination vectors (available from Invitrogen Life Technologies, Carlsbad, Calif.) are constructed for insertion of trait-improving DNA for corn transformation. The elements of each destination vector are summarized in Table 18 below and include a selectable marker transcription region and a DNA insertion transcription region. The selectable marker transcription region comprises a Cauliflower Mosaic Virus 35S promoter operably linked to a gene encoding neomycin phosphotransferase II (nptII) followed by both the 3′ region of the Agrobacterium tumefaciens nopaline synthase gene (nos) and the 3′ region of the potato proteinase inhibitor II (pinII) gene. The DNA insertion transcription region comprises a rice actin 1 promoter, a rice actin 1 exon 1 intron 1 enhancer, an att-flanked insertion site and the 3′ region of the potato pinII gene. Following standard procedures provided by Invitrogen the att-flanked insertion region is replaced by recombination with trait-improving DNA, in a sense orientation for expression of a trait-improving protein and in a gene suppression orientation (i.e., either anti-sense orientation or in a sense- and anti-sense orientation) for a trait-improving suppression of a protein. Although the vector with trait-improving DNA inserted at the att-flanked insertion region is useful for plant transformation by direct DNA delivery, such as microprojectile bombardment, it is preferable to bombard target plant tissue with tandem transcription units that have been cut from the vector. For Agrobacterium-mediated transformation of plants the vector also comprises T-DNA borders from Agrobacterium flanking the transcription units.

Vectors for Agrobacterium-mediated transformation are prepared with each of the trait-improving genes having a sequence of SEQ ID NO:1 through SEQ ID NO:269 with the DNA solely in sense orientation for expression of the encoded, cognate trait-improving protein and in a gene suppression orientation for suppression of the cognate protein. Each vector is transformed into corn callus which is propagated into a plant that is grown to produce transgenic seed for each transgenic event. Progeny plants are self-pollinated to produce seed which is selected for homozygous seed. Homozygous seed is used for producing inbred plants, for introgressing the trait into elite lines, and for crossing to make hybrid seed. The progeny transgenic plants comprising the trait-improving DNA with a sequence of SEQ ID NO: 1 through SEQ ID NO: 269 have one or more improved traits identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic corn including inbred and hybrids are also produced with DNA from each of the identified homologs of DNA of SEQ ID NO: 1 through SEQ ID NO: 269 to provide transgenic seeds and plants which are identified from total transgenic events by screening for the improved agronomic trait. Transgenic corn plants are also produced where the trait-improving DNA is transcribed by each of the promoters from the group selected from, a maize globulin 1 promoter, a maize oleosin promoter, a glutelin 1 promoter, an aldolase promoter, a zein Z27 promoter, a pyruvate orthophosphate dikinase (PPDK) promoter, a soybean 7S alpha promoter, a peroxiredoxin antioxidant (Per1) promoter and a CaMV 35S promoter.

Seed produced by the plants is provided to growers to enable production of corn crops with improved traits associated with the trait-improving DNA.

TABLE 18 FUNCTION ELEMENT REFERENCE DNA insertion Rice actin 1 promoter U.S. Pat. No. 5,641,876 transcription region Rice actin 1 exon 1, intron 1 U.S. Pat. No. 5,641,876 enhancer DNA insertion AttR1 GATEWAY ™ Cloning Technology transcription region Instruction Manual (att - flanked insertin CmR gene GATEWAY ™ Cloning Technology region) Instruction Manual ccdA, ccdB genes GATEWAY ™ Cloning Technology Instruction Manual attR2 GATEWAY ™ Cloning Technology Instruction Manual DNA insertion Potato pinII 3′ region An et al., (1989) Plant Cell 1: 115-122 transcription region selectable marker CaMV 35S promoter U.S. Pat. No. 5,858,742 transcription region nptII selectable marker U.S. Pat. No. 5,858,742 nos 3region U.S. Pat. No. 5,858,742 PinII 3′ region An et al., (1989) Plant Cell 1: 115-122 E. coli maintenance ColEl origin of replication region Fl origin of replication Bla ampicillin resistance

Example 5 Soybean Transformation Construct

Constructs for use in transformation of soybean are prepared by restriction enzyme based cloning into a common expression vector. Elements of an exemplary common expression vector are shown in Table 19 below and include a selectable marker expression cassette and a gene of interest expression cassette. The selectable marker expression cassette comprises Arabidopsis act 7 gene (AtAct7) promoter with intron and 5′UTR, the transit peptide of Arabidopsis EPSPS, the synthetic CP4 coding region with dicot preferred codon usage and a 3′ UTR of the nopaline synthase gene. The gene of interest expression cassette comprises a Cauliflower Mosaic Virus 35S promoter operably linked to a trait-improving gene in a sense orientation for expression of a trait-improving protein and in a gene suppression orientation (i.e., either anti-sense orientation or in a sense- and anti-sense orientation for a trait-improving suppression of a protein.

Vectors similar to that described above are constructed for use in Agrobacterium mediated soybean transformation systems, with each of the trait-improving DNA having a sequence of SEQ ID NO:1 though SEQ ID NO:269 and the respective identified homologs with the DNA in sense orientation for expression of the encoded, cognate protein and in a gene suppression arrangement for suppression of the cognate protein. Each vector is transformed into soybean embryo tissue to produce transgenic events which are grown into plants that produce progeny transgenic plants and seed for screening to identify the transgenic soybean plants of this invention that exhibit the enhanced agronomic trait imparted by DNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respective homolog. The transgenic soybean plants of this invention are identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic soybean plants are also produced where the trait-improving DNA is transcribed by a napin promoter and Arabidopsis SSU promoter.

Seed produced by the plants is provided to growers to enable production of soybean crops with improved traits associated with the trait-improving DNA.

TABLE 19 Function Element Reference Agro transformation B-ARGtu.right border Depicker, A. et al (1982) Mol Appl Genet 1: 561-573 Antibiotic resistance CR-Ec.aadA-SPC/STR Repressor of primers from the ColEl plasmid CR-Ec.rop Origin of replication OR-Ec.oriV-RK2 Agro transformation B-ARGtu.left border Barker, R. F. et al (1983) Plant Mol Biol 2: 335-350 Plant selectable marker expression cassette Arabidopsis act 7 gene McDowell et al., (1996) Plant (AtAct7) promoter with Physiol. 111: 699-711. intron and 5′UTR 5′ UTR of Arabidopsis act 7 gene Intron in 5′UTR of AtAct7 Transit peptide region of Klee, H. J. et al (1987) MGG Arabidopsis EPSPS 210: 437-442 Synthetic CP4 coding region with dicot preferred codon usage A 3′ UTR of the nopaline synthase U.S. Pat. No. 5,858,742 gene of Agrobacterium tumefaciens Ti plasmid Plant gene of interest expression cassette Promoter for 35S RNA from U.S. Pat. No. 5,322,938 CaMV containing a duplication of the −90 to −350 region Gene of interest insertion site Cotton E6 3′ end GenBank accession U30508

Example 6 Cotton Transformation

Vectors similar to that described above for soybean transformation are constructed for use in Agrobacterium mediated cotton transformation systems, with each of the trait-improving DNA having a sequence of SEQ ID NO:1 though SEQ ID NO:269 and the respective identified homologs with the DNA in sense orientation for expression of the encoded, cognate protein and in a gene suppression arrangement for suppression of the cognate protein. Each vector is transformed into cotton embryo tissue to produce transgenic events which are grown into plants that produce progeny transgenic plants and seed for screening to identify the transgenic soybean plants of this invention that exhibit the enhanced agronomic trait imparted by DNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respective homolog. The transgenic cotton plants of this invention are identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic cotton plants are also produced where the trait-improving DNA is transcribed by a napin promoter and Arabidopsis SSU promoter.

Seed produced by the plants is provided to growers to enable production of cotton crops with improved traits associated with the trait-improving DNA. 

We claim:
 1. Transgenic seed for a crop plant having in its genome trait-improving recombinant DNA which expresses a succinate semialdehyde dehydrogenase.
 2. Transgenic seed according to claim 1 wherein transgenic plants grown from said seed exhibit increased yield as compared to control plants.
 3. Transgenic seed according to claim 1 wherein expression of said recombinant DNA provides improved tolerance to water deficit stress as compared to control plants.
 4. Transgenic seed according to claim 1 wherein expression of said recombinant DNA provides improved tolerance to salinity stress as compared to control plants.
 5. Transgenic seed according to claim 1 wherein expression of said recombinant DNA provides improved tolerance to heat stress as compared to control plants.
 6. Transgenic seed of claim wherein said DNA is derived from Agrobacterium tumefaciens.
 7. Transgenic seed according to claim 1, wherein, (a) said crop is susceptible to a yield-limiting environment; and (b) transgenic plants grown from said transgenic seed thrive in said yield-limiting environment.
 8. Transgenic seed according to claim 7, wherein said yield-limiting environment is water deficit stress, heat stress, or high salinity stress.
 9. Transgenic seed of claim 1 wherein said succinate semialdehyde dehydrogenase has an amino acid sequence that is at least 90% identical to a consensus amino acid sequence determined from an alignment of sequences for the succinate semialdehyde dehydrogenase of SEQ ID NO: 442 and the homologs disclosed in Table
 17. 10. A method of facilitating production of a crop comprising providing to a grower of said crop transgenic seed of claim
 1. 11. A method according to claim 10, wherein transgenic plants grown from said seed exhibit increased yield as compared to control plants.
 12. A method according to claim 10, wherein (a) said crop is susceptible to a yield-limiting environment; and (b) transgenic plants grown from said transgenic seed thrive in said yield-limiting environment.
 13. A method of claim 10, wherein said yield-limiting environment is heat stress, water deficit stress or high salinity stress.
 14. Transgenic seed for a crop plant, wherein the genome of said transgenic seed comprises trait-improving recombinant DNA from a gene which expresses a protein having an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group consisting of a consensus amino acid sequence for SEQ ID NO: 270 and homologs thereof disclosed in Table 17 through a consensus amino acid sequence for SEQ ID NO:538 and homologs thereof disclosed in Table
 17. 15. Transgenic seed according to claim 14 wherein transgenic plants grown from said seed exhibit increased yield as compared to control plants.
 16. Transgenic seed according to claim 14, wherein (a) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of water deficit stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 270, 273, 300, 302, 306, 310, 313, 315 through 374, 382, 388, 390 through 392, 397, 402 through 431, 434, 436 through 450, 454, 466, 469, 472, 476, 478, 479, 491, 504 through 538, and homologs thereof disclosed in Table 17; (b) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of heat stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 273, 306, 310, 313, 352 through 372, 407 through 412, 419, 434, 436, 437, 442, 444, 445, 466, 479, 491, 504, 505, 508, 509, 512, 533, and homologs thereof disclosed in Table 17; (c) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of high salinity stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 270, 347, 358, 363, 392, 407, 411, 436, 440, 442-450, 476, 478, 504-538, and homologs thereof disclosed in Table 17; (d) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of cold stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 270 through 316, 352, 353, 360, 361, 363, 368, 373, 382, 383, 389, 398, 402 through 407, 409, 413, 414, 416, 431 through 435, 438, 439, 443, 444, 459, 461, 477, 504 through 508, 510, 514, 521, and homologs thereof disclosed in Table 17; (e) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of reduced nitrogen availability stress and said protein has the function of the protein with amino acid sequence of SEQ ID NO: 316, 351, 375, 389 through 401, 505, and homologs thereof disclosed in Table 17; (f) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of shade stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 293, 300, 307, 316, 370, 373 through 388, 397, 400, 444, 468, 511, 535, and homologs thereof disclosed in Table
 17. 17. A recombinant DNA construct comprising a promoter functional in a plant cell operably linked to trait-improving recombinant DNA from gene for a protein having an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group consisting of a consensus amino acid sequence for SEQ ID NO: 270 and homologs thereof disclosed in Table 17 through a consensus amino acid sequence for SEQ ID NO: 538 and homologs thereof disclosed in Table
 17. 18. A method of facilitating production of a crop comprising providing to a grower of said crop transgenic seed of claim
 14. 19. A method according to claim 18, wherein transgenic plants grown from said seed exhibit increased yield as compared to control plants.
 20. A method according to claim 18, wherein (a) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of water deficit stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 270, 273, 300, 302, 306, 310, 313, 315 through 374, 382, 388, 390 through 392, 397, 402 through 431, 434, 436 through 450, 454, 466, 469, 472, 476, 478, 479, 491, 504 through 538, and homologs thereof disclosed in Table 17; (b) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of heat stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 273, 306, 310, 313, 352 through 372, 407 through 412, 419, 434, 436, 437, 442, 444, 445, 466, 479, 491, 504, 505, 508, 509, 512, 533, and homologs thereof; (c) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of high salinity stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 270, 347, 358, 363, 392, 407, 411, 436, 440, 442-450, 476, 478, 504-538, and homologs thereof disclosed in Table 17; (d) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of cold stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 270 through 316, 352, 353, 360, 361, 363, 368, 373, 382, 383, 389, 398, 402 through 407, 409, 413, 414, 416, 431 through 435, 438, 439, 443, 444, 459, 461, 477, 504 through 508, 510, 514, 521, and homologs thereof disclosed in Table 17; (e) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of reduced nitrogen availability stress and said protein has the function of the protein with amino acid sequence of SEQ ID NO: 316, 351, 375, 389 through 401, 505, and homologs thereof disclosed in Table 17; (f) transgenic plants grown from said seed exhibit increased yield as compared to control plants when said plants are grown in a yield-limiting environment of shade stress and said protein has the function of the protein with an amino acid sequence selected from the group consisting of SEQ ID NO: 293, 300, 307, 316, 370, 373 through 388, 397, 400, 444, 468, 511, 535, and homologs thereof disclosed in Table
 17. 